Genes encoding several poly (ADP-RIBOSE) glycohydrolase (PARG) enzymes, the proteins and fragments thereof, and antibodies immunoreactive therewith

ABSTRACT

The isolation and characterization of cDNAs encoding poly(ADP-ribose) glycohydrolase (PARG) enzymes and the amino acid sequences of PARGs from several species are described. PARG is involved in the cellular response to DNA damage and its proper function is associated with the body&#39;s response to neoplastic disorder inducing agents and oxidative stress. Expression vectors containing the cDNAs and cells transformed with the vectors are described. Probes and primers that hybridize with the cDNAs are described. Expression of the cDNA in  E. coli  results in an enzymatically active protein of about 111 kDa and an active fragment of about 59 kDa. Methods for inhibiting PARG expression or overexpressing PARG in a subject for therapeutic benefit are described. Exemplary of PARG inhibitors are anti-sense oligonucleotides. The invention has implications for treatment of neoplastic disorder, heart attack, stroke, and neurodegenerative diseases. Methods for detecting a mutant PARG allele are also described. Antibodies immunoreactive with PARGs and fragments thereof are described.

CROSS REFERENCE TO RELATED APPLICATION

This Application claims the benefits of US Provisional Applicationnumber 60/083,768, filed May 1, 1998. The entire disclose of USProvisional Application 60/083,768 is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was supported in part by the National Institutesof Health (Grant CA43894). The United States Government may have certainrights in the invention.

TECHNICAL FIELD

The present invention relates to poly(ADP-ribose) glycohydrolases(PARGs) and peptides having poly(ADP-ribose) glycohydrolase activity. Inaddition, the invention also relates to antibodies, including monoclonalantibodies and antibody fragments, that have specific interaction withepitopes present on poly(ADP-ribose) glycohydrolases. Methods oftreatment and diagnosis using the poly(ADP-ribose) glycohydrolases, andantibodies specific for poly(ADP-ribose) glycohydrolases are disclosed.The present invention has implications for the treatment of neoplasticdisorder, reperfusion following ischemia, neurological disorders, andrelated conditions.

BACKGROUND OF THE INVENTION

Genomic damage, if left unrepaired, can lead to malignanttransformation, or cell death by senescence (aging), necrosis orapoptosis. Among the variables that can affect the ultimate biologicalconsequence of DNA damage to a particular cell are (i) the amount, type,and location of the DNA damage and (ii) the efficiency andbioavailability of the cellular DNA repair mechanism.

The activation of poly(ADP-ribose) polymerase (PARP) by DNA strandbreaks is often one of the first cellular responses to DNA damage. PARcatalyzes the conversion of nicotinamide adenine dinucleotide (NAD) tomulti-branched polymers containing up to 200 ADP-ribose residues.Increases in polymer levels of more than 100-fold may occur withinminutes of DNA damage. Once synthesized, polymers are rapidly turnedover, being converted to free ADP-ribose by the action ofpoly(ADP-ribose) glycohydrolase (PARG) (1). An ADP-ribosyl protein lyasehas been proposed to catalyze removal of protein-proximal ADP-ribosemonomers (2). FIG. 1 illustrates these processes schematically.

The process of activating PARP upon DNA damage can rapidly lead toenergy depletion because each ADP-ribose unit transferred by PARPconsumes one molecule of NAD, which in turn, requires six molecules ofATP to regenerate NAD. Additionally, NAD is a key carrier of electronsneeded to generate ATP via electron transport and oxidativephosphorylation or by glycolysis. The overactivation of PARP due tosubstantial DNA damage can significantly deplete the cellular pools ofNAD and ATP (3). ADP-ribose polymer metabolism, and thus PARP and PARGhave been linked to the enhancement of DNA repair (4), limitation ofmalignant transformation (5), enhancement of necrotic cell death (6),and involvement in programmed cell death (7). To date, studies of thestructure and function of the enzymes of ADP-ribose polymer metabolismhave been mainly limited to PARP (8). Little is known about the functionand regulation of PARG.

BRIEF SUMMARY OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to nucleic acids molecules, peptides, methods, vectors andantibodies that are related to the poly(ADP-ribose) glycohydrolase(PARG) enzyme.

One embodiment of the invention is directed to an isolated and purifiednucleic acid molecule or nucleic acid molecule analog comprising asequence that encodes a polypeptide having poly(ADP-ribose)glycohydrolase (PARG) activity. The nucleic acid molecule may encode thecomplete full-length PARG gene or a fragment of the PARG gene. Thenucleic acid molecule may be DNA, RNA or peptide nucleic acid (PNA). Thenucleic acid molecule can be linear, such as, for example, an isolatedfragment or a linear phage DNA. In addition, the isolated nucleic acidmolecule may be circular, such as for example in a plasmid. The nucleicacid molecule may also be a single stranded DNA or RNA such as thesingle stranded DNA or RNA in a single stranded DNA virus or singlestranded RNA virus. The nucleic acid molecule may be of yeast, insect ormammalian origin.

The nucleic acid molecule of the invention, may be of mammalian origin,such as, for example of bovine or murine origin. In a preferredembodiment of the invention, the nucleic acid molecule may be of humanorigin. While the sequence of the nucleic acid molecule is of mammalianorigin, the nucleic acid molecule may be replicated in another organismsuch as an insert in a viral genome, a plasmid in a bacterium or a2-micron plasmid in a yeast.

Preferably, the nucleic acid molecule has, a high degree of sequencesimilarity with a sequence shown in SEQ ID NO: 1 (Genbank AccessionNumber U78975), SEQ ID NO: 3 (Genbank Accession Number AF005043), SEQ IDNO: 5 (Genbank Accession Number AF079557), SEQ ID NO: 7 (GenbankAccession Number AF079556) or SEQ ID NO: 9 (Genbank Accession NumberCEF20C5). The high degree of sequence similarity may be, for example,about 70%, preferably about 80%, even more preferably about 90% and mostpreferably substantially identical such as for example about 100%identity.

The nucleic acid molecule that encodes a polypeptide havingpoly(ADP-ribose) glycohydrolase (PARG) activity may be single or doublestranded nucleic acid molecule of any length such as, for example, about20 bases in length, about 30 bases in length, about 40 bases in length,about 50 bases in length, about 100 bases in length, about 200 bases inlength, about 500 bases in length, about 1000 bases in length, about1500 bases in length, about 2000 bases in length, about 3000 bases inlength. It is understood that “bases” in this patent application means“basepairs” when referring to double stranded nucleic acid molecules andbases when referring to single stranded nucleic acid molecules. In apreferred embodiment of the invention, the nucleic acid molecule may beat least about 1000 base or basepairs long and have at least about 80%sequence similarity with a sequence shown in SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.

In one embodiment of the invention, the nucleic acid molecule may havesequence similarity to one region-of the PARG sequence. The region maybe, for example, from about base residue 2113 to about residue 3105 ofSEQ ID NO: 3. Alternatively, the region may be, from residue 1240 toabout residue 3105 of SEQ ID NO: 3 or from residue 175 to about residue3105 of SEQ ID NO: 3.

Another embodiment of the invention is directed to the expression andoverexpression of PARG in a cell. Expression vectors may mediate theexpression of a polypeptide with poly (ADP-ribose) glycohydrolase (PARG)enzyme activity. Expression systems and expression vectors are known inthe art. For example, one expression vector may comprise a regulatorysequence which is operatively linked to a nucleotide sequence at leastabout 1000 base pairs in length, which has at least 70% sequencesimilarity with a sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. In a preferred embodiment, thesequence similarity is at least about 80% identity, more preferably atleast about 90% identity and most preferably about 100% identity. Theexpression vector may be any expression vector that is capable ofdirecting expression of a gene in ahost cell including, prokaryotic,eukaryotic, mammalian and viral vector. Examples of such vectors includepCMV-Script cytomeglovirus expression vectors for expression inmammalian cells, pESP and pESC vectors for expression in S. pombe and S.cerevesiae, pET vectors for expression in bacteria, pSPUTK vectors forhigh-level transient expression, and pPbac and pMbac vectors forexpression in fall army worm (SF9) cells. Such vectors are availablecommercially from suppliers such as, for example, Invitrogen (Carlsbad,Calif.) or Stratagene (La Jolla, Calif.). In the use of viral vectors,it is understood that defective viral vectors—vectors that aregenetically engineered to deliver a gene or gene product to a host butwhich cannot replicate in a host is preferred. Procedures for thepractice of in vitro and in vivo expression are well known to those ofskill in the art and are further available with the specific expressionproducts and cell lines from commercial suppliers.

Another embodiment of the invention is directed to a host celltransformed with a vector containing a nucleic acid molecule with asequence that encodes a polypeptide having poly(ADP-ribose)glycohydrolase (PARG) activity. The host cell may be any eukaryotic orprokaryotic cell such as, for example a human, murine, rattus, bovine,insect, yeast or bacteria. Specific cell lines are well known to thoseof skill in the art and are available from suppliers such as theAmerican Tissue Type Collection (ATCC, Manassas, Va.) and Stratagene (LaJolla, Calif.) and the like. A preferred embodiment of the invention isdirected to cells transformed with the PARG expression vector whichshows an elevated level of PARG relative to non-transformed cells.Especially preferred are cells transformed with an inducible PARGexpression vector that have normal or slightly elevated PARG levelsbefore induction and have significantly elevated PARG levels afterinduction.

An embodiment of the invention is directed to an isolated protein havingpoly(ADP-ribose) glycohydrolase (PARG) activity. The protein maycomprise an amino acid sequence with at least 70% sequence similaritywith a sequence shown in SEQ ID NO: 2 (Genbank Accession Number U78975),SEQ ID NO: 4 (Genbank Accession Number AF005043), SEQ ID NO: 6 (GenbankAccession Number AF079557), SEQ ID NO: 8 (Genbank Accession NumberAF079556), or SEQ ID NO: 10 (Genbank Accession Number CEF20C5). Thesequence similarity is preferably at least about 80%, more preferably atleast about 90% and most preferably substantially identical with asequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, or SEQ ID NO: 10. In a preferred embodiment of the invention, thepreferred isolated protein having poly(ADP-ribose) glycohydrolase (PARG)activity and has a molecular weight greater than about 100 kDa.

Another embodiment of the invention is directed to an oligonucleotidewhich is greater than about 10 bases in length and less than about 1000bases in length which is complementary to a sequence shown SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Theoligonucleotide may be, for example, greater than about 20 bases inlength, greater than about 30 bases in length, greater than about 40bases in length, greater than about 50 bases in length, greater thanabout 100 bases in length, greater than about 200 bases in length orgreater than about 300 bases in length. The oligonucleotide, which maybe optionally labeled with a detectable marker, may be DNA, RNA or PNA.A detectable marker may be, for example, a radioactive isotope such as32p or 1251, an epitope such as FLAG.

One preferred oligonucleotide is an antisense oligonucleotide directedto the mRNA of PARG. Antisense oligonucleotide as a method ofsuppression is well known to those in the art. For example, thephosphorothioate oligonucleotide, ISIS 2922, has been shown to beeffective against cytomeglovirus retinitis in AIDS patients (9). It isthus well known that oligonucleotides, when administered to animals andhumans, can have a useful therapeutic effect. In a preferred embodiment,the oligonucleotide is at least about 10 nucleotides in length, such as,greater than about 20 bases in length, greater than about 30 bases inlength, greater than about 40 bases in length, greater than about 50bases in length, greater than about 100 bases in length, greater thanabout 200 bases in length or greater than about 300 bases in length. Inanother preferred embodiment, the oligonucleotide has a ribozymeactivity.

Another embodiment of the invention is directed to an isolatedpolypeptide of at least 6 amino acid residues in length and having amolecular weight less than about 65 kDa, which has at least about 80%sequence similarity with a sequence shown in any one of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. Thepolypeptide may be, for example, at least about 10 amino acids inlength, at least about 20 amino acids in length, at least about 30 aminoacids in length, at least about 40 amino acids in length, at least about50 amino acids in length, at least about 75 amino acids in length, atleast about 100 amino acids in length, at least about 150 amino acids inlength, at least about 250 amino acids in length or at least about 500amino acids in length or more.

In a preferred embodiment, the polypeptide has a molecular weight lessthan about 40 kDa and has at least about 90% sequence similarity with asequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8 or SEQ ID NO: 10. The polypeptide preferably haspoly(ADP-ribose) glycohydrolase (PARG) activity or is immunogenic andelicits antibodies immunoreactive with a poly(ADP-ribose) glycohydrolase(PARG) enzyme. In a more preferred embodiment, the polypeptide comprisesan amino acid sequence substantially identical with SEQ ID NO: 4 fromabout residue 647 to about residue 977.

Another embodiment of the invention is directed to an isolatedpolypeptide of at least 10 amino acid residues in length and which hasat least about 80% sequence similarity with a sequence shown in any oneof SEQ ID-NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO:10. Preferably, the polypeptide is at least about 20 amino acids inlength, such as, for example at least about 30 amino acids, about 40amino acids, about 50 amino acids, about 100 amino acids, about 200amino acids and about 500 amino acids in length.

Another embodiment of the invention is directed to an antibodyimmunoreactive with an isolated polypeptide of at least about 6 aminoacid residues in length and having a molecular weight less than about 65kDa, which has at least about 80% sequence similarity with a sequenceshown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8 or SEQ ID NO: 10. In a preferred embodiment, antibody isimmunoreactive with a polypeptide with a molecular weight less thanabout 40 kDa and has at least about 90% sequence similarity with asequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8 or SEQ ID NO: 10. In another preferred embodiment, theantibody is immunoreactive with a polypeptide comprising an amino acidsequence substantially identical with SEQ ID NO: 4 from about residue647 to about residue 977.

Another embodiment of the invention is directed to a method of detectinga polypeptide having PARG activity comprising the steps of contactingthe polypeptide with an antibody immunoreactive with an isolatedpolypeptide of at least about 6 amino acid residues in length and havinga molecular weight less than about 65 kDa, which has at least about 80%sequence similarity with a sequence shown in any one of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, anddetermining whether the antibody immunoreacts with the polypeptide.

Another embodiment of the invention is directed to a method ofpreventing, treating, or ameliorating a disease condition or disorder inan individual comprising the step of administering a therapeuticallyeffective amount of a poly(ADP-ribose) glycohydrolase (PARG) inhibitoror activator to the individual. The disease condition or disorder may beany condition associated with responses to DNA damage, examples of whichinclude a neoplastic disorder, a myocardial infarction, a vascularstroke or a neurodegenerative disorder. The PARG inhibitor or activatormay be a small molecule inhibitor or activator of PARG or may be anantisense oligonucleotide that can hybridize in vivo to messenger RNAencoded by a PARG gene. PARG based treatment may be directed to newmethods for preventing, treating or ameliorating disorders associatedwith DNA damage. These disorders include neoplastic disorders, inborngenetic errors, myocardial infarctions, vascular strokes, aging, andneurodegenerative disorders such as Alzheimer's disease, Huntington'sdisease, Parkinson's disease, and neurotoxicity generally.

Another embodiment of the invention is directed to the identification ofnovel PARG modulators which can activate or inhibit DNA repair and/orapoptosis. A PARG modulator is a compound that can activate or inhibitPARG. These modulators are preferably more efficacious and do not havethe known side effects of present modulators. One method of identifyingan agent that inhibits or activates poly(ADP-ribose) glycohydrolase(PARG) activity comprise the steps of providing a liquid medium thatcontains a polypeptide having PARG activity contacting the polypeptidewith a candidate agent, in the presence of a reference compound havingaffinity for the polypeptide, under predetermined assay conditions, anddetermining the affinity of the candidate agent for the polypeptiderelative to the reference compound. Thus, the modulation activity of thecandidate agent relative to the reference compound is determined. Inthis method, the polypeptide may be immobilized on a solid support.Further, the polypeptide may be generated in vitro by culturing a celltransformed with a nucleic acid molecule encoding PARG under conditionseffective to express the polypeptide.

Another embodiment of the invention is directed to a method ofidentifying a mutant PARG allele in an individual comprising the step ofobtaining genomic material from the individual; digesting the genomicmaterial with a restriction enzyme having a recognition site inclusiveof the mutant allele; fractionating the restriction fragments obtainedfrom the digestion; and comparing the fractionation pattern with thatobtained for a normal allele, thereby determining the presence orabsence of the mutant allele. The fractionating step may be performedwith electrophoresis.

Another embodiment of the invention is directed to a method ofidentifying a mutant PARG allele in an individual comprising the stepsof hybridizing an oligonucleotide with genomic material from theindividual, which oligonucleotide hybridizes under predeterminedhybridization conditions to a region immediately 5′ of a predeterminedmutation site in the PARG alleles with the 3′ terminus of theoligonucleotide complementary to an unmutated PARG allele; extending theoligonucleotide using PCR amplification; and determining the degree towhich extension occurs, thereby determining the presence or absence ofthe mutant allele. The PCR extension reaction may be performed at atemperature above about 50° C. The determination may be performed byconducting electrophoresis (using for example, acrylamide at about 4% toabout 10% or agarose and low melting temperature agarose from about 0.8%to about 4%) on the products of PCR amplification.

Another embodiment of the invention is directed to a method of screeningmolecules for PARG modulating activity (inhibition or activation)comprising the steps of providing a purified PARG enzyme; assaying theenzyme in the presence of a molecule to be screened; and comparing theactivity of the PARG enzyme in the presence of the molecule to theactivity of the PARG enzyme in the absence of the molecule.

Another embodiment of the invention is directed to a method of genetherapy comprising the step of delivering an oligonucleotide having asequence complementary to at least a portion of a polynucleotideencoding a PARG enzyme to a cell to be treated. In the method, theoligonucleotide may have a sequence complementary to a sequence encodinga C-terminal portion of a PARG enzyme. Further, in the gene therapymethod, the oligonucleotide may further comprise a ribozyme.

Another embodiment of the invention is directed to a method ofdelivering to a cell surface, an oligonucleotide having a sequencecomplementary to at least a portion of a polynucleotide encoding a PARGenzyme to a cell to be treated. In the method, the oligonucleotide mayhave a sequence complementary to a sequence encoding a C-terminalportion of a PARG enzyme. Further, in the method, the oligonucleotidemay further comprise a ribozyme. The portion of a polynucleotideencoding a PARG enzyme may be, for example, the polynucleotide encodingthe N terminus third of PARG, the middle third of PARG, or the Cterminus third of PARG. The portion of a polynucleotide may encode asmaller part of PARG such as the N terminus 10% of PARG, the C terminus10% of PARG, or any 10% portion in between such as from 10% to 20%, from20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60%to 70%, from 70% to 80%, from 80% to 90%. The percent value used means apercent of the linear amino acid sequence. Thus, for a 1000 amino acidprotein, the N terminus 10 percent is from amino acid 1 to 100; 10% to20% percent would be from amino acid 100 to 200 and so on. For a 970amino acid protein, the N terminal 10% would be from amino acid 1 to 97;10% to 20% would be from amino acids 98 to 194 amino acids.

Another embodiment of the invention is directed to a method ofsensitizing a cell to a chemotherapeutic agent comprising the step ofcontacting the cell with a molecule that modulates the activity of aPARG enzyme. The molecule may be an oligonucleotide having a sequencecomplementary to at least a portion of a polynucleotide encoding a PARGenzyme. For example, the oligonucleotide may have a sequencecomplementary to a sequence encoding a C-terminal portion of a PARGenzyme. The portion of a polynucleotide encoding a PARG enzyme may be,for example, the polynucleotide encoding the N terminus third of PARG,the middle third of PARG, or the C terminus third of PARG. The portionof a polynucleotide may encode a smaller part of PARG such as the Nterminus 10% of PARG, the C terminus 10% of PARG, or any 10% portion inbetween such as from 10% to 20%, from 20% to 30%, from 30% to 40%, from40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80%to 90%. The oligonucleotide may further comprise a ribozyme. The methodmay be used, for example, as a method of treating a diseased cellcharacterized by the presence of DNA strand breaks. In the treatment,the cell is contacted with a molecule that modulates an enzymaticactivity of a PARG enzyme.

Another embodiment of the invention is directed to a pharmaceuticalcomposition comprising an oligonucleotide having a sequencecomplementary to at least a portion of a polynucleotide encoding a PARGenzyme. The produced molecule may be an oligonucleotide having asequence complementary to at least a portion of a polynucleotideencoding a PARG enzyme. For example, the oligonucleotide may have asequence complementary to a sequence encoding a C-terminal portion of aPARG enzyme. The oligonucleotide may comprise a ribozyme activity.

Another embodiment of the invention is directed to a virus that causesthe production of an oligonucleotide having a sequence complementary toa polynucleotide encoding a PARG enzyme. This may be, for example, aviral vector which after the infection of a host cell, causes theproduction of an antisense RNA of PARG. The molecule may be anoligonucleotide having a sequence complementary to at least a portion ofa polynucleotide encoding a PARG enzyme. For example, theoligonucleotide may have a sequence complementary to a sequence encodinga C-terminal portion of a PARG enzyme. The oligonucleotide may furthercomprise a ribozyme activity.

Other embodiments and advantages of the invention are set forth, inpart, in the description that follows and, in part, will be obvious fromthis description and may be learned from the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cellular biochemical process that occurs after DNAdamage.

FIG. 2 depicts the SDS-PAGE analysis of purified bovine thymus PARG.

FIG. 3. depicts the alignment of the DNA sequences of two PCR productsand eight λgt11 cDNA clones used to identify the cDNA coding for bovinePARG.

FIG. 4 depicts a northern blot analysis of bovine kidney cells mRNAtranscripts.

FIG. 5 depicts an alignment of the putative bipartite NLS of bovine,human, and murine PARG and comparison with the bipartite NLS of PARPfrom different organisms.

FIG. 6 depicts expression of bPARG enzyme activity in E. coli (10).

FIG. 7 depicts a Southern blot analysis of bovine DNA probed with PARGcDNA.

FIG. 8 depicts activity gel autoradiogram of E. coli expressed bovinePARG.

FIG. 9 depicts the analysis by anion exchange HPLC of material releasedfrom ADP-ribose polymers by PARG action.

FIG. 10 depicts the SDS-PAGE analysis of the purification of E. coliexpressed GST-PARG.

FIG. 11 depicts a schematic representation of the portions of the bovinePARG cDNA expressed as GST fusion constructs.

FIG. 12 depicts the cloning of the 1.8 kb PCR EcoRI fragment encodingfor the 65 kDa catalytic domain of PARG.

FIG. 13 depicts an autoradiogram of an activity gel of GST-PARG fusionconstructs expressed in E. coli and PARG expressed in baculovirus.

FIG. 14 depiots a schematic representation of the strategy used toisolate cDNA molecules encoding PARG from various organisms.

FIG. 15 depicts the domain organization of PARGs from differentorganisms.

FIG. 16 depicts an amino acid sequence alignment of bovine, murine,human, drosophila and C. elegans PARG enzymes.

FIG. 17 depicts a western blot of recombinant PARGs.

FIG. 18 depicts western blots of natural and recombinant expressed PARG.

FIG. 19 depicts the characterization of PARG by Western Blot in mousecells of different PARP genotypes.

FIG. 20 depicts a partial restriction map of the mouse PARG locus.

FIG. 21 depicts a schematic representation of the strategy used tocreate PARG knockout mice.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS LIST OF ABBREVIATIONS

-   ADP adenosine diphosphate-   ADPR ADP-ribose-   AMP adenosine monophosphate-   ASPCR allele-specific PCR-   bp base pair(s)-   bPARG bovine PARG-   CePARG C elegans PARG-   dPARG Drosophila melanogaster PARG-   DTT dithiothreitol-   GSH-Sepharose Glutathione-Sepharose 4B-   GST glutathione-S transferase-   hPARG human PARG-   HPLC high pressure liquid chromatography-   ICE interleukin-1 b converting enzyme-   IPTG isopropyl-β-D-thiogalactoside-   kb kilobase pair(s)-   MDBK Madin-Darby bovine kidney cells-   mPARG murine PARG-   NAD nicotinamide adenine dinucloetide-   NLS nuclear location signal-   PADPR DHB-Sepharose poly(ADP-ribose)-dihydroxyboronyl-Sepharose-   PAGE polyacrylamide-gel electrophoresis-   PARG poly(ADP-ribose) glycohydrolase-   PARP poly(ADP-ribose) polymerase [EC 2.4.2.30]-   PCR polymerase chain reaction-   PEG-6,000 polyethylene glycol 6,000-   PEG polyethylene glycol-   PMSF phenylmethylsulfonyl fluoride-   PR-AMP phosphoribosyl-adenosine monophosphate-   RFLP restriction fragment length polymorphism-   SDS sodium dodecyl sulfate-   SSCP single-strand conformation polymorphism-   TPCK Trypsin: L-1-tosylamido-2-phenylethyl chloromethyl ketone.

An “agonist” as defined herein refers to a molecule which, when bound toPARG, increases or prolongs the effect of PARG. Agonist may includeproteins, nucleic acid molecules, carbohydrates, or any other moleculesthat bind to and modulate the effect of PARG.

An “allele” or “allelic sequence”, as defined herein refers to analternative form of PARG. Alleles may result from at least one mutationin the nucleic acid molecule sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles, aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

An “ortholog” as defined herein refers to a nucleotide or amino acidsequence that is related to a reference nucleotide or amino acidsequence through speciation, and is therefore identical or structurallysimilar to the reference sequence.

A given nucleotide or amino acid sequence is said to be “substantiallyidentical” with another sequence when the compared sequences have thesame residues in the same order, excepting for any degeneracy(nucleotides) and conservative substitutions (amino acids).

A “regulatory sequence” of an expression vector is a DNA sequencenecessary for inducing transcription of a gene, and includes afunctional promoter and/or enhancer sequence. The term “operativelylinked” as used herein means that a first nucleotide sequence, such as aregulatory element, is fused in frame with a second nucleotide sequenceso as to afford a faithful transcription of the entire nucleotidesequence, which upon translation yields the desired protein.

The term “immunoreactivity” and related terms refers to the ability ofantibodies and fragments thereof to bind to particular regions(antigens) presented by polypeptides and proteins, presented to theantibodies either as immunogens or targets. Typically, the bindingaffinity of the antibodies for their antigen is in the range 10⁵ to10¹¹, with higher affinities being preferred.

The term “specific immunoreactivity” refers to the ability of antibodiesand fragments thereof to bind to particular regions (antigens) presentedby polypeptides and proteins, presented to the antibodies either asimmunogens or targets and not to unrelated antigens. For example, anantibody with specific immunoreactivity to actin will bind actin butwould not bind another protein, such as a polymerase, which do not shareepitopes with actin.

The term “nucleic acid molecule” refers to DNA, RNA and nucleic acidmolecule analogs such as PNA and the like. PNA or “Peptide Nucleic Acid”is a nucleic acid molecule analog that has a neutral “peptide-like”backbone with nucleobases that allow the molecule to hybridize tocomplementary RNA or DNA with higher affinity and specificity thancorresponding oligonucleotides. PNA can be made to be more resistant tonormal nucleases and are especially desirable, for example, in genetherapy. PNA is known to one of skill in the art and can be purchased orcustom synthesized in numerous commercial laboratories includingPerSeptive Biosystems, Inc. (Framingham, Mass.).

The term “modulate” means to activate or inhibit. For example, a PARGmodulator may activate or inhibit PARG activity. “Modulation activity”means the amount of activation or inhibition. For example, a compoundthat increase PARG (or any other enzyme) activity by. 10% will have amodulation activity of 10%. Conversely, a compound that decreases PARGactivity by 10% will have a modulation activity of −0%.

As used herein, a given nucleotide or amino acid sequence is said tohave a defined percentage of sequence similarity with another sequencewhen the two sequences differ by no more than the specified sequencesimilarity, including conservative substitutions, insertions, anddeletions. Degenerate codons do not result in a change in amino acidupon translation, therefore, it is appreciated that identical aminoacids-can be encoded by several equivalent codons. The term “homology”and “sequence similarity” should have the same meaning for the purposeof this patent. Similarity parameters may be any generally acceptableparameter. For the purposes of this patent, percent similarity betweentwo polymers such as nucleic acid molecules and polypeptides ispreferably defined by Karlin and Altschul (11). The similarityalgorithms of Karlin and Altschul are well known to those of skill inthe art as exemplified by their adoption by the National Center forBiological Information. For nucleic acid molecule sequence searching,one desirable set of parameters would M (score for a pair of matchingresidues) at 5; N (score for mismatching residues) at −4; W (wordlength) at 11. For proteins, it is well known that some amino acids aresimilar and that substitution would be conservative. That is, forexample, the replacement of an acidic amino acid with another acidicacid would be consider a conservative mismatch while the replacement ofan acidic amino acid with a basic amino acid would be consider a moredivergent mismatch. Preferably, the parameters for a desirable proteinsimilarity determination are expressed in the sequence similarity matrixBLOSUM62 as described in Henikoff & Henikoff (12). Other similaritymatrixes that are also preferred in the invention are PAM40, PAM120 andPAM250 as described in Altschul (13).

The rapid synthesis of ADP-ribose polymers that occurs in response toDNA strand breaks is accompanied by very rapid polymer turnover,indicating that PARP and PARG activities are closely coordinated ascells respond to DNA damage. While PARP has been widely studied,information concerning structure and function relationships of PARG ismuch more limited. The present invention discloses the isolation of acDNA encoding the bovine, human, murine and drosophila PARG and theirdeduced amino acid sequences.

The availability of PARP cDNA has allowed a number of molecular geneticapproaches to study the function(s) of ADP-ribose polymer metabolism andthe availability of PARG cDNA should allow the design of additionalmolecular genetic approaches for studying this metabolism. For example,disruption of the gene encoding PARG in mice containing a normal PARPgene will allow the determination of whether other cellular enzymes canreplace PARG in the turnover of ADP-ribose polymers and/or whetherdevelopment of animals will occur in the absence of PARG. Alternatively,disruption of the PARG gene in mice containing a disrupted PARP gene mayprovide insights for the coordinated function of PARP and PARG.

One embodiment of the invention is directed to a deoxyribonucleic acid(DNA) molecule that encodes a polypeptide having poly(ADP-ribose)glycohydrolase (PARG) activity. Preferably, the molecule is of mammalianorigin, such as, for example, of human origin.

In a preferred embodiment, a DNA molecule of the invention comprises anucleotide sequence with at least about 70% sequence similarity with asequence shown in a sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. Higher degrees of sequencesimilarity, such as about 80%, about 90%, and about 100% are preferred.Most preferred is a DNA molecule comprising a nucleotide sequencesubstantially identical with any one of sequence shown in SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. It ispreferred that a DNA molecule of the present invention comprises atleast about 1000 nucleotides and has a nucleotide sequence with at least80% sequence similarity with a sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. Most preferably, the DNAmolecule consist of a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,and SEQ ID NO: 9.

For a DNA molecule of the present invention based on a human PARG geneit is preferred that the molecule comprises a nucleotide sequence thatshows similarity to the sequence shown in SEQ ID NO: 3 from aboutresidue 2113 to about residue 3105. More preferably, the sequencesimilarity is from about residue 1240 to about residue 3105. Still morepreferably, the DNA molecule comprises a nucleotide sequence similarityto the coding sequence for the full-length hPARG as shown in SEQ ID NO:3 from about residue 175 to about residue 3105.

A DNA molecule of the present invention affords probes and primermolecules that can be used in hybridization assays and PCRamplification. An exemplary oligonucleotide is less than about 1000residues in length and comprises a nucleotide sequence at least about 10residues long to ensure hybridization. Preferably, the at least about 10residue region of the oligonucleotide is complementary to a sequenceshown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQID NO: 9. Typically, the oligonucleotide will be a DNA molecule, whichcan be labeled by any method as desired, for example, with a radiolabel,a fluorescence label, or chemi-luminescent label.

Another embodiment of the invention is directed to a nucleic acidmolecule that hybridizes to in a nucleic acid blot (Southern blot,Northern blot) to a sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7, or SEQ ID NO: 9 under stringent hybridizationconditions. A nucleic acid blot may be made using techniques defined inMolecular Cloning, Second Edition, Sambrook et al., Cold Spring HarborPress, Cold Spring Harbor, N.Y. DNA to be analyzed may be separated inagarose or acrylamide gels. The DNA may be transferred to nylon ornitrocellulose membrane using techniques known to those in the art.Stringent hybridization condition may be for example, prehybridizations42° C. in 50% formamide, 0.25 M sodium phosphate buffer, pH 7.2, 0.25 MNaCl, 7% SDS, 1 mM EDTA for 10 hours, 100 ug denatured salmon sperm DNA,hybridization at 42° C. in 50% formamide, 0.25 M sodium phosphatebuffer, 100 ug denatured salmon sperm DNA, pH 7.2, 0.25 M NaCl, 7% SDS,1 mM EDTA, 1 ng/ml probe with a specific activity of 10⁹ cpm/ug DNA, for16 hours. The probe may comprise any contiguous sequence from SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.Preferably, said contiguous sequence is at least about 50 bases long,more preferably, the contiguous sequence is at least about 75 baseslong, such as at least about 100 bases, at least about 200 bases long orat least about 300 bases long. The complete sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Methods oflabeling probes to with radioactive labels are known to those of skillin the art.

Method of washing after stringent hybridization are known. A stringentwashing may comprise, for example, two washes at in 2×SSC, 0.1% SDS. for15 minutes each at room temperature; two washes in 0.2×SSC, 0.1% SDS for15 minutes each at room temperature; and a final three washes in0.2×SSC, 0.1% SDS for 15 minutes each at 60° C. The final wash may beincreased in temperature for reduced background. For example, the finalwash may be a final three washes in 0.2×SSC, 0.1% SDS for 15 minuteseach at 65° C. or a final three washes in 0.2×SSC, 0.1% SDS for 15minutes each at 68° C.

If a radioactive probe is used, hybridization may be monitored usingknown techniques such as autoradiogram or a two dimensional measurementof radioactivity.

An anti-sense oligonucleotide is also afforded by the present invention.The anti-sense molecule is typically less than about 1000 residues inlength to ensure ease of synthesis, and hybridizes to an RNA molecule,e.g., messenger RNA, which has at least 70% sequence similarity with asequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9. Preferably, the anti-sense molecule is at least about 10nucleotides in length to ensure hybridization with mRNA. Even morepreferably, the anti-sense molecule may be at least about 15 nucleotidesin length such as, for example, at least about 20 nucleotides in length;at least about 30 nucleotides in length; at least about 50 nucleotidesin length; at least about 75 nucleotides in length; at least about 100nucleotides in length; at least about 150 nucleotides in length; atleast about 200 nucleotides in length; at least about 500 nucleotides inlength; at least about 1000 nucleotides in length; or at least about1500 nucleotides in length. It is also preferred that the molecule has aribozyme activity so that it can degrade the mRNA that it binds to.

An antisense oligonucleotide may be used therapeutically to inhibittranslation of mRNA encoding PARG. Synthetic antisense oligonucleotidesmay be produced, for example, in a commercially availableoligonucleotide synthesizer. This invention provides a means totherapeutically alter levels of expression of a human or other mammalianPARG by the use of a synthetic antisense oligonucleotide drug thatinhibits translation of mRNA encoding PARG. Synthetic antisenseoligonucleotides, or other antisense chemical structures designed torecognize and selectively bind to mRNA, are constructed to becomplementary to portions of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9. Anantisense oligonucleotide may be designed to be stable in the bloodstream for administration to patients by injection, or in laboratorycell culture conditions, for administration to cells removed from thepatient. The antisense may be designed to be capable of passing throughcell membranes in order to enter the cytoplasm and nucleus of the cellby virtue of physical and chemical properties of the antisenseoligonucleotide which render it capable of passing through cellmembranes (e.g., by designing small, hydrophobic antisenseoligonucleotide chemical structures) or by virtue of specific transportsystems in the cell which recognize and transport the antisenseoligonucleotide into the cell. In addition, the antisenseoligonucleotide can be designed for administration only to certainselected cell populations by targeting the antisense oligonucleotide tobe recognized by specific cellular uptake mechanisms which bind and takeup the antisense oligonucleotide only within certain selected cellpopulations. For example, the antisense oligonucleotide may be designedto bind to transporter found only in a certain cell type, asdiscussed-above. The antisense oligonucleotide may be designed toinactivate the PARG mRNA by (1) binding to the PARG mRNA and thusinducing degradation of the mRNA by intrinsic cellular mechanisms suchas RNase I digestion, (2) by inhibiting translation of the mRNA targetby interfering with the binding of translation-regulating factors or ofribosomes, or (3) by inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups, which either degrade orchemically modify the target mRNA. Synthetic antisense oligonucleotidedrugs have been shown to be capable of the properties described abovewhen directed against mRNA targets (14). In addition, coupling ofribozymes to antisense oligonucleotides is a promising strategy forinactivating target mRNA (15). In this manner, an antisenseoligonucleotide directed to PARG may serve as a therapy to reduce PARGexpression in particular target cells of a patient and in any clinicalcondition that may benefit from reduced expression of PARG.

It is known by those in the art that as a result of the degeneracy ofthe genetic code, a multitude of nucleotide sequences encoding PARG,some bearing minimal homology to the nucleotide sequences of any knownand naturally occurring gene, may be produced. Thus, the inventioncontemplates a nucleic acid molecule that encodes a polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8 and SEQ ID NO:10. The invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices that would encode the oligopeptides disclosedherein. These combinations are made in accordance with the standardtriplet genetic code as applied to the nucleotide sequence of naturallyoccurring PARG and all such variants are to be considered as beingspecifically disclosed.

Although nucleic acid molecules which encode PARG and its variantspreferably hybridizes under high stringency conditions to the nucleotidesequence of the naturally occurring PARG gene under appropriateconditions of stringency, it may be advantageous to produce nucleotidesequences encoding PARG or its derivatives possessing a substantiallydifferent codon usage. Codons may be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding PARG and its derivatives andvariants without altering the produced amino acid sequence include theproduction of RNA transcripts having more desirable properties, such asgreater half-life, than transcripts produced from the naturallyoccurring sequence.

In order to express a biologically active or immunologically activePARG, the nucleic acid molecule encoding PARG or functional equivalents,may be inserted into appropriate expression vector, such as, for examplea vector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Thus, another aspect of thepresent invention is an expression vector comprising a regulatorysequence operatively linked to nucleic acid molecule comprising anucleotide sequence disclosed herein. For example, an expression vectorcan contain a nucleotide sequence at least about 1000 base pairs inlength, which has at least about 70%, about 80%, or higher, sequencesimilarity with a sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.

Methods that are known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding PARG andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination.

A variety of expression vector/host systems may be utilized to containand express sequences encoding PARG. These include, for example,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus), insects infected withvirus expression vectors (e.g., fall army worm infected withbaculovirus); plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus;TMV) or with bacterial expression vectors (e.g., Ti or bacterialplasmids); or animal cell systems. The invention is not limited by thehost cell employed.

Prokaryotic expression systems are commercially available from a numberof suppliers worldwide. Prokaryotic expression vectors provide aconvenient system to synthesize proteins. If it is desired to express aprotein with characteristics such as immunogenic properties, 3Dconformation, and other features exhibited by authentic PARG, theprotein may be expressed in an eukaryotic protein expression system. Theeukaryotic expression systems are numerous and include mammalian,amphibian, plant, insect, and yeast expression systems.

Yeast hosts that can be used for expression include Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastois, Hanselapolymorpha, Kluyveromyces lactis, and Yarrowia lipolytica. Yeast hostsoffer the advantages of rapid growth on inexpensive minimal media andease in large-scale production using bioreactors. Another advantage ofyeast is the ability to direct expression to cytoplasmic localization orfor extracellular export.

Most yeast vectors for protein expression are derivatives of the S.cerevisiae 2μ (two micron) plasmid. Yeast vectors include pYES and pESTfrom Stratagene (La Jolla, Calif.). Constitutive gene expression by theyeast plasmid cassette can be mediated by well known promoters such asthe glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3); the triosephosphate isomerase promoter (TPI1); the phosphoglycerate isomerasepromoter (PGK1); the alcohol dehydrogenase isozyme II (ADH2) genepromoter; GAL1 and GAL10 promoters; the metallothionein promoter fromthe CUP1 gene (induced by copper sulfate); and the PHO5 promoter(induced by phosphate limitation). Proper termination of yeasttranscripts is known to those in the art. Termination signals mayinclude the MF-alpha-1, TP11, CYC1, and PGK1 genes. These terminationsignals may be spliced onto the 3′ end of the insert to provide propertermination.

Insect expression systems include baculovirus based vectors designed toexpress foreign proteins in a number of insect hosts and insect cellline hosts. Insect and insect cell lines may be of Drosophilamelanogaster, Aedes albopictus, Spodoptera frugiperda, and Bombyx moriorigin. Numerous expression systems comprising cells, vectors, hosts andthe like can be purchased from a variety of commercial sources.

The control elements or regulatory sequences necessary for the properexpression of the insert, in this case PARG, may comprises promoters,enhancers (including both proximal and distal control elements) whichinteract with the host proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificityand are known to those in the art. Depending on the vectors system andhost utilized, any number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used.For example, the LacZ promoter may be used in a bacterial cell; thebaculovirus polyhedrin promoter may be used in an insect cell; plantpromoters such as heat shock promoters, and storage protein promoters,plant virus promoters and the like may be used in a plant cell. In amammalian cell expression system, an SV40 promoter or EBV promoter maybe used.

Methods and protocols for both prokaryotic and eukaryotic expressionsystems are generally known to those in the art. Further, the cells,vectors, growth medium may be purchased from commercial suppliers. Thecatalogs and product literature of commercial suppliers provide detailedprotocols to enable the expression of proteins in prokaryotic andeukaryotic systems including bacterial, yeast, insect, insect cell, andmammalian cell systems. The product literature and catalogs of Clontech(Palo Alto, Calif.), Invitrogen (Carlsbad, Calif.), Life Technologies(Rockville, Md.), Novagen (Madison, Wis.), Pharmigen (San Diego,Calif.), Quantum Biotechnotogies (Montreal, Quebec, Canada), andStratagene (La Jolla, Calif.) are incorporated herein by reference.

A further aspect of the invention is isolated proteins and proteinfragments having poly(ADP-ribose) glycohydrolase (PARG) activity. Such aprotein can comprise an amino acid sequence with sequence similarity ofat least about 70%, about 80% or higher to a sequence shown SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. Forexample, the full-length bovine PARG has a molecular weight greater thanabout 100 kDa, thereby distinguishing it from previously known PARGs.The protein may be purified, for example, from cell lysates using theantibodies of the invention. The purification may be through an antibodycolumn.

PARG polypeptides are another aspect of the invention. Polypeptides ofPARG may be used, for example, to generate antibodies in an immunogenicprocedure. To be effective it is preferred that the polypeptides are atleast about 6 amino acid residues in length, such as for example, atleast about 10 amino acids in length, at least about 20 amino acids inlength, at least about 30 amino acids in length, at least about 50 aminoacids in length, at least about 75 amino acids in length, at least about100 amino acids in length, at least about 150 amino acids in length, atleast about 200 amino acids in length, or at least about 400 amino acidsin length. In one embodiment, the polypeptide has a molecular weightless than about 65 kDa and with at least about 80% sequence similaritywith a sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, and SEQ ID NO: 10. The polypeptide may consist of the sequenceset forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, orSEQ ID NO: 10.

The polypeptide of the invention may be conjugated to a larger molecule,such as, for example, keyhole lymphet hemocyanin (KLH), to increase theimmunogenicity of the polypeptide. The increased immunogenicity of thepolypeptide will, in turn, increase the yield of antibody. Preferably,the polypeptide has a molecular weight less than about 40 kDa and withat least about 90% sequence similarity with a sequence shown in SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. Thepolypeptide can also be used in a wide variety of assays, e.g., as acompetitor of antigen in a liquid sample in an antibody-based assay.Therefore, it is preferred that the polypeptide has poly(ADP-ribose)glycohydrolase (PARG) activity. A particularly preferred polypeptide isof human origin and comprises an amino acid sequence substantiallyidentical with SEQ ID NO: 4 from about residue 647 to about residue977—the C terminus catalytic region of the enzyme. Longer sequences moreinclusive of the natural molecule are of course also contemplated.

The invention also encompasses PARG variants and alleles. A preferredPARG variant is one having at least 80% and more preferably at least 90%amino acid similarity to the amino acid of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 and which retains at leastone biological, immunological or other functional characteristic oractivity of PARG. A most preferred PARG variant is one having at least95% amino sequence similarity or identity to human PARG (SEQ ID NO: 3).

Antibodies to PARG may be generated using numerous established methodsthat are well known in the art. One example of such a method isdescribed in the Examples. Generated antibodies may include, forexample, polyclonal, monoclonal, chimeric, single chain, Fab fragments,Fab′ fragments, Fab′(2) fragments, and fragments produced by a FABexpression library. Humanized antibodies and single chain antibodies mayalso be produced after the amino acid sequence of effective antibodiesare determined.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith PARG or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, for example,Freund's mineral gels such as aluminum hydroxide, and surface-activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Amongadjuvant used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to PARG have an amino acid sequence consisting of atleast five amino acids and more preferably at least about 10 aminoacids, such as for example about 20 amino acids or about 40 amino acids.It is also preferable that they are identical to a portion of the aminoacid sequence of the natural PARG. Short stretches of PARG amino acidsmay be fused with those of another protein such as keyhole limpethemocyanin and antibodies may be produced against the chimeric molecule.

Antibodies may be produced by inducing in vivo production in thelymphocyte population of a living animal or by screening immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inpublished procedures (16).

Antibody fragments that contain specific binding sites for PARG may begenerated. For example, such fragments include the F(ab′)₂ fragment, Fabfragment, Fab′ fragment which can be produced by enzymatic digestion ofthe antibody molecule. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse, W. D. (1989) Science 254,1275-1281).

Therapeutic Methods

A method of preventing, treating, or ameliorating a disease condition ina patient, which disease state is affected by the level of PARGexpression is also contemplated. This method entails administering atherapeutically effective amount of a poly(ADP-ribose) glycohydrolase(PARG) inhibitor or activator to the individual. Particularly,implicated disease states are neoplastic disorder, myocardialinfarction, vascular stroke and neurodegenerative disorders.

In one embodiment, antisense oligonucleotides for PARG may be used aloneor in combination with other chemotherapeutic agents to treat neoplasticdisorder. The anti-sense oligo is designed to hybridize in vivo tomessenger RNA expressed by the organism. The use of anti-sense moleculesin a therapeutic setting is described, for example, by S. Agrawal,Antisense Therapeutics, Humana Press. Currently favored protocols callfor the oligo to have ribozyme activity in an effort to degrade themRNA. These methods are described, for example, in TherapeuticApplication of Ribozymes, K. Scanlon, ed., Humana Press. Therefore, inone embodiment, an antagonist of PARG may be administered to a subjectto prevent or treat neoplastic disorder.

PARG levels may be enhanced to suppress DNA repair and increase a cell'ssusceptibility to chemotherapy drugs. Therefore, in another embodiment,an PARG enhancer is administered to a subject along with achemotherapeutic drug as a treatment for neoplastic disorder.

Neoplastic disorders that can be treated by PARG elevation andchemotherapy include benign and malignant neoplasm such as, for example,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, hyperplasia and hypertrophy. Neoplastic disorders mayinclude, in particular, neoplastic disorders of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. For the purposes of thisinvention, a neoplastic disorder is any new and abnormal growth;specifically a new growth of tissue in which the growth is uncontrolledand progressive. Malignant cancer is a subset of neoplastic disorderswhich show a greater degree of anaplasia and have the properties ofinvasion and metastasis.

The synthesis of effective anti-sense inhibitors is known. Numerousapproaches have been previously described and generally involve alteringthe backbone of the polynucleotide to increase its stability in-vivo.Exemplary oligonucleotides and methods of synthesis are described inU.S. Pat. Nos. 5,661,134; 5,635,488; and 5,599,797 (phosphorothioatelinkages), U.S. Pat. Nos. 5,587,469 and 5,459,255 (N-2 substitutedpurines), U.S. Pat. No. 5,539,083 (peptide nucleic acids) and U.S. Pat.Nos. 5,629,152; 5,623,070; and 5,610,289 (miscellaneous approaches). Thedisclosures of each of these references are incorporated herein byreference.

Significantly, the present invention discloses a method of identifyingan agent that inhibits or activates poly(ADP-ribose) glycohydrolase(PARG) activity. Such method comprises (i) providing a liquid mediumthat contains a polypeptide of the present invention; (ii) contactingthe polypeptide with a candidate agent, in the presence of a referencecompound having affinity for the polypeptide, under predetermined assayconditions; and (iii) determining the affinity of the candidate agentfor the polypeptide relative to the reference compound, therebydetermining the inhibition or activation activity of the candidate agentrelative to the reference compound. These determinations can befacilitated by immobilizing the polypeptide on a solid support.Alternatively, the polypeptide can be generated in vitro by culturing acell transformed with a PARG gene under conditions effective to expressthe polypeptide.

Combination therapies are also afforded by the present invention inwhich a PARG inhibitor or activator is administered in combination witha chemotherapeutic or a “clot-busting” drug. The clot-busting drug maybe, for example, tissue plasminogen activator (t-PA) or streptokinase.

In some cases it may be desired to overexpress PARG in the cells of anorganism in order to achieve the correct PARP/PARG balance. In thiscontext of gene therapy, it is desired to stably transfect target cellswith a vector, such as, for example, a viral or a DNA (nucleic acid)vector, so that the desired gene is overexpressed. Gene therapy vectorsystems and protocols are well known and are described, for example, inthe Internet Book of Gene Therapy (]7) Anti-sense and ribozymeapproaches to cancer gene therapy are described in chapters 7-9 of theInternet Book of Gene Therapy, and are incorporated herein by reference.Another reference is Gene Therapy Protocols, P. Robbins, ed., HumanaPress. Furthermore, gene therapy methods have advanced greatly and arewell documented in numerous issued U.S. patents. Gene therapy may bepracticed, for example, by substituting a nucleic acid molecule of theinvention with the nucleic acid molecule described in the methodsreferred to in any issued U.S. patents directed to gene therapy (18).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods

Methods of genotyping an individual for a mutant PARG allele are alsoafforded by the present invention. A number of protocols are availablefor identifying a mutant allele as described herein once the nucleotidesequence encoding PARG is known. Some exemplary methods are restrictionfragment length polymorphism (RFLP), allele-specific PCR (ASPCR) andsingle-strand conformation polymorphism (SSCP). Armed with thisinformation, the genetic susceptibility of an individual to anabove-mentioned disease condition can be assessed.

An allele-specific method for identifying point mutations bydifferential PCR amplication is described by (19). A non-electrophoreticmethod of genotyping with allele-specific PCR employs a dye specific fordouble-stranded DNA (20). A method of detecting mutations referred to assingle-stranded conformation polymorphism (SSCP) is presently widelyemployed (21). A hybrid of SSCP and Sanger dideoxy sequencing, calleddideoxy fingerprinting (ddF) has recently been described (22).

Other methods of identifying allelic mutations are known to the skilledartisan. Probably the most commonly used method of genotyping isrestriction fragment length polymorphism (RFLP) (23), which is employsone or more restriction enzymes to identify mutant alleles occurringwithin a restriction site. This method has been used extensively inforensic applications and is employed commercially by such companies asHelix Biotech, Inc. Reliagene Technologies, Inc. and GenTestLaboratories, Inc. Accordingly, an instant mutant PARG allele can bedetected by RFLP methods, optionally by one of these commercialentities. The above methods are most effective in the detection ofhomozygotes for the defective allele.

An RFLP method of identifying a mutant PARG allele in an individualentails: (i) obtaining genomic material from the individual; (ii)digesting the genomic material with a restriction enzyme having arecognition site inclusive of the mutant allele; (iii) fractionating therestriction fragments obtained from the digestion, e.g., byelectrophoresis; and (iv) comparing the fractionation pattern with thatobtained for a normal allele, thereby determining the presence orabsence of the mutant allele.

An ASPCR method of identifying a mutant PARG allele in an individualentails: (i) hybridizing an oligonucleotide with genomic material fromthe individual; (ii) attempting to extend the oligonucleotide using PCRamplification; and (iii) determining the degree to which extensionoccurs, thereby determining the presence or absence of the mutantallele. In this method, it is preferred that the oligonucleotidehybridizes under predetermined hybridization conditions to a regionimmediately 5′ of a predetermined mutation site in the PARG allele withthe 3′ terminus of the oligonucleotide complementary to an unmutatedPARG allele. In these protocols, the PCR extension reaction is generallyattempted at a temperature above about 50° C., more preferably aboveabout 60° C.

A variety of protocols including ELISA, RIA and FACS for measuring PARGlevels are known in the art and provide a basis for diagnosing alteredor abnormal levels of PARG expression. Normal or standard values forPARG expression may be established by combining body fluids and tissuebiopsies from normal mammalian subjects, rupturing the cells orpermeating the cells, combining the cells with antibody under conditionssuitable for complex formation. The amount of standard complex formationmay be quantified by various methods but preferably by photometricmeans. Quantities of PARG expressed in subject, control, and diseasesample are compared to standard values to determine between normal,reduced or enhanced levels of PARG.

A still further aspect of the invention pertains to an antibodyimmunoreactive with a polypeptide of the present invention. Preferablythe antibodies are specifically immunoreactive with the polypeptides ofthis invention such as, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, and SEQ ID NO: 10. Frequently it is desired to label theantibody, e.g., with a radiolabel, fluorescent or epitope label, topermit visualizing the antibody. Thus, antibodies immunoreactive withthe PARG of this invention are afforded, which can be used to studyfeatures of PARG heterogeneity and possible modes of regulation. Thehigh degree of sequence similarity between bovine PARG, human PARG andmurine PARG permits eliciting antibodies to PARG of one species, whichare found to be cross-reactive with PARGs from other organisms. Theseantibodies are valuable in characterizing PARG in-vivo under definedphysiological conditions in many different organisms.

Accordingly, a method of detecting a polypeptide having PARG activity,for example, a diagnostic assay, entails: (i) contacting the polypeptidewith an aforementioned antibody of the invention; and (ii) determiningwhether the antibody immunoreacts with the polypeptide. Binding can beascertained in an sandwich assay, as is well known, due to the abilityof the antibodies to immunoreact with an epitope of PARG. Preferably,monoclonal antibodies, such as those prepared by the method of Kohlerand Milstein (24) and labeled antigens effective in competing with thepolypeptide, are employed. Exemplary assays are disclosed in U.S. Pat.No. 4,375,110, the disclosure of which is incorporated herein byreference.

The present invention includes immunoreactive fragments of a PARGenzyme. Immunoreactive fragments can be fragments that can elicit animmune response that recognizes a PARG enzyme. Alternatively,immunoreactive fragments can be fragments that are specifically bound byan antibody that specifically binds a PARG enzyme. Any of variety ofmethods may be employed in order to identify contiguous peptidefragments of a PARG enzyme that comprise immunoreactive sequences. PARGenzymes may be fractionated by proteases, cyanogen bromide, etc. and theresultant fragments assessed for their capacity to specifically bindanti-PARG antibodies.

In an alternative embodiment, one or more synthetic peptides may beprepared in order to locate contiguous amino acid sequences that areimmunoreactive. The peptides may have a sequence that includes a seriesof contiguous amino acids that are identical to a series of contiguousamino acids of a PARG enzyme. The peptides may be of about six aminoacids to about 500 amino acids in length. The peptide may also includesequences that are not identical to sequences of a PARG so long as itincludes at least about six contiguous amino acids that are identical toabout six contiguous amino acids of a PARG enzyme. In a preferredembodiment, the peptide will be about 50 amino acids in length. In otherpreferred embodiments the length of the peptide may be from about sixamino acids to about 30 amino acids.

The peptides of the present invention may comprise amino acid sequencesthat elicit antibodies that specifically bind to the peptide or to aPARG enzyme. Alternatively, the peptides may contain sequences that arespecifically bound by anti-PARG antibodies. Peptides that are bound byanti-PARG antibodies may identified through the use of Epitope Scanning™strategy (Cambridge Research Biochemicals, Inc.). Thus, the linearsequence of amino acids of a particular PARG enzyme is used to constructa set of peptides of defined length which overlap other members of theset by one or more residues. The peptides may be any length; however,lengths of from about 6 to 25 amino acids are preferred. In selectingthe length, a general consideration is that antibodies that recognizelinear native epitopes constitute approximately 60-70% of theanti-protein antibody population (25).

The number of overlapping amino acids will generally be more than halfof the length of the peptides. That is, if the peptides are about 20amino acids long, the overlap may be 11 or more amino acids long. Inpreferred embodiments, each peptide will be selected such that thenumber of overlapping amino acid residues in adjacent peptides is fromabout (n-1) to (n-3), where “n” is the number of amino acids in thepeptide. An overlap of (n-I) is particularly preferred. Thus, in aparticularly preferred embodiment, a first peptide may have the aminoacid sequence of residues 1-10 of a PARG enzyme, a second peptide mayhave the amino acid sequence of residues 2-11 of the same PARG enzyme, athird peptide may have the sequence of residues 3-12 of the same PARGenzyme and so on until the entire sequence of the PARG enzyme has beensynthesized in fragments.

The peptides may be synthesized using any means known to those of skillin the art. In a preferred embodiment, the peptide will be synthesizedusing an automated synthesizer such as a multipin peptide synthesissystem. Such systems or peptides synthesis services are commerciallyavailable from suitable providers known to those skilled in the art.

To identify suitable peptides, each peptide is introduced into a well ofa microtiter plate, and assayed for its ability to bind to antibodieselicited by a PARG enzyme. Such assays may be conducted in various waysknown to those skilled in the art. One suitable assay is conducted byimmobilizing a peptide on the surface of a well and then contacting thepeptide with a solution containing an anti-PARG antibody. After washing,the well is contacted with a labeled antibody that specifically binds tothe anti-PARG antibody. Thus, the presence of label in the wellindicates that the anti-PARG antibody bound to the immobilized peptide.Another preferred method of determining the ability of the peptide to bespecifically recognized by anti-PARG antibodies is a competitive ELISA.

Once a particular peptide has been found to bind to anti-PARGantibodies, the peptide can be used to elicit monospecific antibodies.By immunizing an experimental animal with a single peptide containing asingle antigenic determinant, the antibodies elicited will allspecifically bind to the same antigenic determinant even though theantibodies are not monoclonal.

Where desired, the peptides can be modified to increase theirimmunogenicity. Thus, they may be modified to contain an amino-terminaland/or a carboxyl-terminal cysteine or lysine residue with or withoutspacer arms. The peptides may be conjugated to carriers such as bovineserum albumin, ovalbumin, human serum albumin, KLH (keyhole limpethemocyanin) or tetanus toxoid. The use of human serum albumin ispreferred over ovalbumin or bovine serum.

The peptides, alone or conjugated to a carrier, may be themselvescapable of eliciting an antibody response when administered to anexperimental animal. Alternatively, the peptides, alone or conjugated toa carrier, may be administered in conjunction with an adjuvant. Thoseskilled in the art will understand that a variety of materials mayfunction as adjuvants. Examples of possible adjuvants include, but arenot limited to, Freund's complete adjuvant, Freund's incompleteadjuvant, lipopolysaccharide (LPS) and the like. Any material thatincreases the immune response to a fragment of a PARG enzyme may be usedas an adjuvant.

The ability to produce large amounts of active PARG enzyme permits, forthe first time, the large scale screening of chemical libraries formolecules capable of inhibiting or activating PARG enzymatic activity.The screening may be conducted using any assay for PARG known to thoseskilled in the art. In a preferred embodiment, the screen may beconducted using the TLC based assay described by Ménard, et al. (26). Aknown amount of PARG will be incubated under standardized conditionswith [³²P]-poly(ADPR) in the presence of inhibitor or activator. Afteran appropriate period of time, the reaction will be stopped and thereaction mixture separated on PEI-F cellulose TLC plates. The TLC platesmay be developed in an appropriate solvent system such as methanolfollowed by 0.3N LiCl. The amount of ADPR released in the reaction willbe quantified and the effect of the inhibitor or activator on enzymaticactivity will be determined. Typical reaction conditions are 50 mMpotassium phosphate (pH 7.5) at 37° C. in the presence of 25 μM[³²P]-poly(ADPR). The concentration of the inhibitor or activator can bevaried as necessary to determine the K_(i) value of the inhibitor oractivator according to standard procedures.

Another embodiment of the invention is directed to a method of alteringthe response of the cell to a genotoxic stress by modulating theconcentration of ADPR polymers. As discussed above, the metabolism ofADPR polymers is critical in determining the fate of cells subjected togenotoxic stress. The modulation can be either an increase or a decreasein the concentration of the polymers. In one embodiment of the presentinvention, the concentration of ADPR polymers can be decreased by theuse of a gene therapy vector expressing a high level of PARG. In anotherembodiment of the present invention, the concentration of polymers canbe increased by inhibiting the enzymatic activity of the PARG enzyme bythe addition of inhibitors or activators identified as described above.Alternatively, the concentration of ADPR polymers can be increased byinterfering with the endogenous expression of PARG enzymes usingantisense oligonucleotide technology.

Knowledge of the nucleotide sequence of the PARG gene permits thepreparation of antisense therapeutics containing sequences complimentaryto the mRNA of PARG gene. The preparation and delivery of antisensetherapeutics is well known to those skilled in the art. For example,antisense therapeutics have been used to treat neoplastic disorder asexemplified by Smith, U.S. Pat. No. 5,248,671, specifically incorporatedherein by reference. Additional examples of antisense therapeutics areprovided by Miller, U.S. Pat. Nos. 4,511,713 and 4,757,055, specificallyincorporated herein by reference.

In the present invention, an oligonucleotide having a sequencecomplimentary to the mRNA of the PARG gene will be prepared. Such anoligonucleotide is said to be an antisense oligonucleotide with respectto the PARG gene. The oligonucleotide may be RNA or DNA or a may containboth RNA and DNA portions. The oligonucleotide may contain modifiedbonds so as to enhance the stability of the oligonucleotide and renderit more resistant to the action of cellular nucleases. For example, theoligonucleotide may be constructed with phosphorothioate nucleotides,phosphonate nucleotides and other types of modified nucleotides known tothose skilled in the art. The structure of the oligonucleotide may bealtered so as to include other types of bonds that do not naturallyoccur in oligonucleotides. For example, adjacent nucleosides might bejoined using linear alkyl chains, peptide bonds or other types ofstructures. The only limitation is that the resulting oligonucleotideremains capable of hybridizing to the target PARG mRNA.

The antisense oligonucleotides may be delivered by any means customarilyused in the art. For example, the oligonucleotide may be delivered inneutral liposomes, cationic liposomes or by ballistic high speedinjection. Alternatively the DNA sequence encoding the antisenseoligonucleotide may be inserted into a gene vector and the vector may beintroduced into target cells. The vector may be any type of gene therapyvector known to those skilled in the art. Preferred embodiments include,plasmid vectors and viral vectors. Viral vectors are seen to includethose vectors customarily used for gene therapy applications including,but not limited to; retroviral vectors, vaccinia virus vectors, herpesvirus vectors, adenovirus vectors and adeno-associated virus vectors.Upon introduction of the vector into target cells, the vector willdirect expression of a nucleic acid molecule comprising the appropriatesequence to hybridize with the mRNA encoding a PARG enzyme. In apreferred embodiment, introduction of the vector into the target cellwill result in the production of an RNA molecule that hybridizes withthe mRNA of a PARG enzyme and also includes one or more additional RNAsequences capable of functioning as a ribozyme. The ribozyme portion ofthe molecule will cause the cleavage of the mRNA encoding the PARGenzyme thereby preventing the production of PARG.

Therapeutics of this type may be used to treat a wide variety ofconditions. In one embodiment, an antisense therapeutic will be used totreat neoplastic disorder. In a preferred embodiment, an antisensetherapeutic of the present invention will be delivered in combinationwith a currently known chemotherapeutic agent. In general,chemotherapeutic agents function by disrupting the integrity of DNA intarget cells. Since the recovery of a cell from such DNA disruption ishighly dependent upon the normal ADPR polymer metabolism, the presenceof the antisense therapeutic will have the effect of chemosensitizingthe neoplastic cells by disturbing the ratio PARG and PARP.

In another preferred embodiment, the antisense oligonucleotides of thepresent invention may be used to treat a variety of conditions caused bygenotoxic oxidative stress. Examples include cardiac disorders, neuronaldisorders, reperfusion injury, neurotoxicity, Alzheimer's disease,Huntington's disease and Parkinson's disease. It has been shown thatinhibition of ADPR polymer synthesis provides protection againstcellular damage caused by nitric oxide injury. Zhang, et al,. U.S. Pat.No. 5,587,384, specifically incorporated herein by reference, teach thatdecreasing the amount of ADPR polymers formed can result in protectionagainst nitric oxide induced neurotoxicity. As discussed above,decreasing the amount of ADPR polymers in the cell can be accomplishedby the introduction of gene therapy vector expressing PARG, thus, thepresent invention can be used to treat neurodegenerative conditionsresulting from oxidative stress.

CONCLUSION

The synthesis and rapid turnover of ADP-ribose polymers is an immediatecellular response to DNA damage. Reported here is the isolation andcharacterization of cDNAs encoding various poly(ADP-ribose)glycohydrolase (PARG) enzymes responsible for ADP-ribose polymerturnover. PARG was isolated from bovine thymus, yielding a protein ofapproximately 59 kDa. Based on the sequence of oligopeptides derivedfrom the enzyme, polymerase chain reaction products and partial cDNAclones were isolated and used to construct a putative full-length cDNA.The cDNA of approximately 4.1 kb pairs predicts expression of a proteinof approximately 111 kDa, nearly twice the size of the isolated protein.A single transcript of approximately 4.3 kb pairs is detected in bovinekidney poly(A)⁺ RNA, consistent with expression of a protein of 111 kDa.Expression of the cDNA in Escherichia coli results in an enzymaticallyactive protein of 111 kDa and an active fragment of 59 kDa. Analysis ofrestriction endonuclease fragments from bovine DNA by Southernhybridization indicate that PARG is encoded by a single copy gene. Takentogether, the results indicate that previous reports of multiple PARGscan be explained by proteolysis of an 111-kDa enzyme. The deduced aminoacid sequence of the bovine PARG shares little or no sequence similaritywith differing types of known proteins; however, it contains a putativebipartite nuclear location signal as would be predicted for a nuclearprotein. The availability of cDNA clones for PARG should facilitatestructure-function studies of the enzyme and its involvement in cellularresponses to genomic damage.

Other embodiments and advantages of the invention are set forth, inpart, in the description that follows and, in part, will be obvious fromthis description and may be learned from practice of the invention.

EXAMPLES Example 1 Purification of Bovine PARG

PARG was purified from bovine thymus tissue (Pel-Freez, Rogers, A K) bymodifications of previously published procedures (27). The enzyme wasisolated up to the polyethylene glycol (PEG)-6,000 fractionation step asdescribed previously (28). However, DNA-agarose and heparin-Sepharosechromatographic steps used previously were omitted, and the PEG-6,000fraction was applied directly to an affinity matrix ofpoly(ADP-ribose)-dihydroxyboronyl-Sepharose (PADPR DHB-Sepharose). Theactive fractions eluted from PADPR DHB-Sepharose (25 ml) were pooled,placed in dialysis tubing, concentrated against dry PEG-20,000 toapproximately 12 ml, and dialyzed against 2 liters of 20 mM potassiumphosphate buffer, pH 8.0, 0.1% Triton X-100, 5 mM β-mercaptoethanol, 0.1mM thioglycolic acid, 0.4 M KC1 (buffer A). The sample was loaded onto a1.0×11-cm Toyopearl AF-Red (Supelco) column, and PARG was eluted with an80-ml linear gradient of 0.4-2 M KCl in buffer A. The active fractions,eluting at approximately 1.25 M KCl, were pooled, placed in dialysistubing, concentrated against solid sucrose to approximately 9 ml, anddialyzed against 20 mM potassium phosphate buffer, pH 7.2, 0.75 M KCl,0.1% Triton X-100, 10% glycerol, 5 mM β-mercaptoethanol, 0.1 mMthioglycolic acid. PARG activity was determined as described by Menardand Poirier (29), and protein content was determined by the method ofBradford (30). The final preparation was quantified by SDS-PAGE (31) andCoomassie Blue staining to compare the intensity of the protein bandwith a known amount of bovine serum albumin (32).

The purification procedure for the bovine thymus PARG summarized inTable 1 is typical for results obtained from six separate preparationsof the enzyme. Purification from 500 g of bovine thymus achievedapproximately 50,000-fold purification and yielded approximately 20 μgof purified protein. An aliquot of the purified enzyme was precipitatedwith trichloroacetic acid, washed with acetone, resuspended in SDS-PAGEsample buffer, separated on a 10% SDS-PAGE gel, and stained withCoomassie Blue. Analysis of the final preparation of SDS-PAGE revealedthat more than 95% of the protein migrated at an apparent molecular massof approximately 59 kDa (FIG. 2). In FIG. 2, an aliquot of the purifiedenzyme was precipitated by TCA, washed with acetone, resuspended inSDS-PAGE sample buffer, separated on a 10% SDS-PAGE gel and stained withCoomassie blue. The positions of molecular weight marker proteins areshown. TABLE 1 Purification of PARG from bovine thymus Specific Totalactivity Protein activity units/mg Yield Purification Step mg unitsprotein % -fold Crude extract 27,800 57,400 2.06 100 1.0 Protamine12,500 58,000 4.64 101 2.3 sulfate Ammonium 4,480 30,000 6.70 52 3.3sulfate CM-Sepha- 171 19,100 112 33 55 rose PEG 6000 23.0 7,530 327 13160 PADPR-DHB- 1.30 6,730 5,180 12 2,500 Sepharose Toyopearl 0.023 2,26098,300 4 48,000 AF-Red

Example 2 Peptide Sequencing

Prior to proteolytic fragmentation, the purified bPARG (40 μg in 100 μlof 0.4 M ammonium bicarbonate buffer, pH 8.0, 8 M urea) was incubated ina final concentration of 2.2 mM dithiothreitol at 56° C. for 15 min.Iodoacetamide was added to a final concentration of 2.0 mM, and thesample was incubated at 25° C. for 15 min. After dilution with an equalvolume of water, 1.5 units of immobolized L-1-tosylamido-2-phenylethylchloromethyl ketone-treated trypsin (Pierce Chemical, Rockford, Ill.)was added, and the sample was incubated at 37° C. for 18 h with gentlerotary shaking. Finally, the mixture was subjected to centrifugation at16,000×g for 5 min to separate the tryptic fragments from theimmobolized trypsin. The tryptic fragments were adjusted to 0.05% intrifluoroacetic acid and separated on a 4.6 mm×25 cm, Microsorb MV, C₄reversed-phase HPLC column (Rainin) eluted with an 80-min lineargradient from 4 to 44% acetonitrile in 0.05% trifluoroacetic acid. Fouroligopeptide fractions, with approximate elution times of 61, 63, 68,and 75 min, were selected for peptide sequence analysis by the Edmandegradation method. Amino acid sequence data of four oligopeptides,designated by their approximate HPLC elution times from thereversed-phase column, are shown in Table II. TABLE II Amino acidsequence of oligopeptides derived from bPARG Oligopeptide Amino AcidSequence SEQ ID NO: 10 20 30 68 LFTEVLDHNE CLIITGTEQY SEYTGYAETY R SEQID NO:11 63 AYCGFLRPGV SSENLSAVAT GNXGCGAFG SEQ ID NO:12 61 FLINPELIVS RSEQ ID NO:13 75 IALXLPNIXT QPIPLL SEQ ID NO:14

Example 3 cDNA Cloning

To obtain cDNA clones encoding bovine PARG, PCR amplificationexperiments were followed by the screening of two different bovine cDNAlibraries. FIG. 3 depicts the alignment of the DNA sequences of two PCRproducts and eight λ gt11 cDNA clones used to identify the cDNA codingfor bovine PARG. The two PCR products and clones 1 and 2 were obtainedfrom the bovine thymus cDNA library. Clones 3-8 were obtained from thebovine kidney cDNA library. The positions of restriction sites used inthis study are shown, and the top diagram shows the consensus clone,denoting the relative location of the coding regions for oligopeptides,75, 61, 68, and 63 as well as the open reading frame and noncodingregions. For each of the cDNA inserts characterized, the sequence ofboth strands was determined by the dideoxynucleotide chain terminationmethod using Sequenase™ (U.S. Biochemical Corp., Cleveland, Ohio).

The first step leading to the isolation of cDNA clones was to synthesizetwo multi-degenerate 17-mer primers, GAYCAYAAYGARTGYYT (SEQ ID NO: 15)and CKRTANGTYTCNGCRTA (SEQ ID NO: 16) (where Y represents T/C, R is A/G,K is T/G, and N is A/T/C/G), based on two regions of the SEQ ID NO: 11;“DHNECL” (amino acids 7 to 12 of SEQ ID NO: 11) and “YAETYR” (amino acid26 to amino acid 31 of SEQ ID NO: 11) (Table II). Using themultidegenerate primers and an oligo(dT)-primed bovine thymus cDNA λgt11library BL1019b from Clontech (Palo Alto, Calif.), PCR amplificationgenerated a 74-bp DNA fragment with a deduced amino acid sequenceidentical to the corresponding region of oligopeptide 68. Next, twospecific 24-mer oligonucleotide primers, ATCATCACAGGTACTGAGCAGTAC (SEQID NO: 17) and GCCTGTGTATTCACTGTACTGCTC (SEQ ID NO: 18), based on thesequence of this 74-bp DNA were used in combination with λgt11 forwardand reverse primers to amplify PCR products 1 and 2 from the bovinethymus library. PCR product 1 contained 231 bp of sequence including theregion encoding the N-terminal region of oligopeptide 68 (SEQ ID NO: 11)and the entire sequence of oligopeptide 61 (SEQ ID NO: 13). PCR product2 contained 757 bp, which included a sequence encoding the C-terminalregion of oligopeptide 68 (SEQ ID NO: 11) and the entire sequence ofoligopeptide 63 (SEQ ID NO: 12).

The sequence information obtained from PCR products 1 and 2 was used toisolate cDNA clones obtained by the screening of bovine thymus andbovine kidney cDNA libraries. A 518-bp EcoRI-HindIII fragment from PCRproduct 2 was used as a probe to screen approximately 1×10⁶ independentclones from the bovine thymus library. Two positive cDNA clones (clones1 and 2) were isolated, which overlapped PCR products 1 and 2. However,attempts to obtain clones from the bovine thymus library that containedsequence 5′ to clone 2 were unsuccessful. Thus, a 231-bp EcoRI-KpnIfragment from clone 2 was used as a probe to screen approximately 5×10⁵independent clones of the bovine kidney 5′ stretch plus cDNA λgt11library BL300 lb (Clontech, Palo Alto, Calif.). Three positive cDNAclones (clones 3-5) were obtained, all of which contained sequence 5′ toclone 2. Each of these clones also contained a sequence encodingoligopeptide 75. Clones 1-5 provided multiple overlapping sequences inthe 3′-terminal portion of a consensus cDNA, but additional clones weresought to obtain overlapping sequences for the 5′-terminal region. Thus,a 436-bp EcoRI-KpnI fragment located at the 5′ end of clone 3 was usedas a probe to screen approximately 6×10⁵ independent clones of thebovine kidney library. Clones 6-8 provided overlapping sequences for the5′-terminal region. The full-length cDNA was constructed by ligating a3.9-kb XbaI-NsiI fragment from pWL11 l (clone 1 cDNA insert in pTZ18R(33)) and a 3.0-kb NsiI-XbaI fragment from pWL13 (clone 4 cDNA insert inpTZ 18R). The resulting plasmid, termed pWL30, contained the 4,070-bpfull-length cDNA.

FIG. 3 shows an alignment of the DNA sequences of two PCR products andeight λgt11 cDNA clones used to identify the cDNA coding for bovinePARG. The two PCR products and Clones 1 and 2 were obtained from thebovine thymus cDNA library. Clones 3 through 8 were obtained from thebovine kidney cDNA library. The position of restriction sites used inthis study is shown and the top diagram shows the consensus clone,denoting the relative location of the coding regions for oligopeptides75, 61, 68, and 63 as well as the open reading frame and non codingregions.

The nucleotide sequence of cDNA coding for bovine PARG is shown in thesequence listing as SEQ ID NO: 1. The deduced amino acid sequence of theenzyme is shown in the sequence listing as SEQ ID NO: 2. The fouroligopeptides sequenced from purified enzyme is within SEQ ID NO: 2.They are IALCLPNICTQPIPLLK (amino acid 601 to 617, SEQ ID NO: 2);LINPELIVSR (amino acid 761 to 770, SEQ ID NO: 2);LFTEVLDHNECLIITGTEQYSEYTGYAETYR (amino acid 771 to 801, SEQ ID NO: 2)and AYCGFLRPGV PSENLSAVAT GNWGCGAFGGDAR (amino acid 849 to 880, SEQ IDNO: 2). The combined nucleotide sequence of Clones 1 through 8 predicteda full-length cDNA clone of 4,070 bp containing 257 bp of 5 ′-non-codingsequence, a single open reading frame of 2,931 bp (beginning at the ATGat position 258 of SEQ ID NO: 1) and a 3′-non-coding region of882 bp.and the deduced amino acid sequence which predicts a protein of 977amino acids and a molecular weight of 110.8 kDa.

Example 4 Analysis of the Sequence of Bovine PARG

The cDNA clone (SEQ ID NO: 1) has features typical of cDNAs that codefor mammalian proteins. These include (i) an oligo A (putative poly(A)+)sequence at the 3′-end, (ii) a polyadenylation signal (AATAAA) 12 bpupstream from the oligo A sequence, (iii) a sequence of ATTTA in the3′-untranslated region thought to play a role in selective mRNAdegradation in mammalian cells (34), (iv) a single open reading frame,and (v) a nucleotide sequence around the first start codon commonlyfound at known sites of initiation of translation (35). The evidencethat the cDNA clone constructed represents a full-length or nearlyfull-length clone for PARG is shown by the observation thathybridization of poly(A)+ RNA from bovine kidney cells with the cDNAshowed a single band of hybridization of approximately the same size asthe cDNA under stringent hybridization conditions (set forth above)(FIG. 4).

The nucleotide sequence encoding bovine PARG indicates that PARG shareslittle or no sequence similarity with other known sequences. A search ofsequence data banks has failed to reveal significant sequence similaritywith any sequences coding for known proteins. A strong sequencesimilarity has been observed with human and rat cDNA clones that likelyrepresent partial clones for PARG from these species. Examination ofprotein sequence databases such as Genbank and SwissPro also has shownthat the deduced amino acid sequence of PARG lacks any sequencesimilarity with known proteins. However, the amino acid sequence sharesa significant similarity with a protein sequence from Caenorhabditiselegans that may represent the PARG protein from this organism (36).

The deduced amino acid sequence of PARG has been examined for a numberof structural motifs that can be predicted from the primary amino acidsequence. The expressed PARG protein was observed to be able to formdimers stable to SDS-PAGE conditions. In that regard, residues 871-907show significant homologies to known leucine zipper dimerizationsequences (37).

Another motif identified is a putative bipartite nuclear location signal(NLS) (38). It is interesting that PARP also contains a bipartite NLS(39). FIG. 5 compares deduced amino acid sequences in the NLS region ofthe bovine PARG, and regions of putative PARG sequences from human,mouse and C. elegans, with the NLS region of PARP from seven differentorganisms. Conserved residues are noted in bold and the amino aciddistances are from the amino terminal methionine residue. Abbreviationsand references for the sequences shown are as follows: bPARG, bovinePARG (SEQ ID NO: 19); hPARG, human PARG (SEQ ID NO: 20); mPARG, murinePARG (SEQ ID NO: 21); CePARG, Caenorhabditis elegans PARG (SEQ ID NO:22); hPARP, human PARP (SEQ ID NO: 23; 40); mPARP, murine PARP (SEQ IDNO: 24; 41); bPARP, bovine PARP (SEQ ID NO: 25, 42); aPARP, chicken PARP(SEQ ID NO: 26; 43); XIPARP, Xenopus laevis PARP (SEQ ID NO: 27; 44);DmPARP, Drosophila melanogaster PARP (SEQ ID NO: 28; 45); SpPARP,Sarcophaga peregrina PARP (SEQ ID NO: 29; 46). In FIG. 5, conservedresidues are noted in boldface type, and the amino acid distances arefrom the amino-terminal methionine residue. Sequence alignment ofputative bipartite nuclear localization signal of bovine, human andmurine PARG compared to the nuclear localization signal of PARP fromdifferent organisms. The putative NLS of PARG fulfills the criteria forbipartite NLS in that it contains conserved acidic and basic amino acidresidues at two different locations each within the region of sequencesimilarity to the NLS of PARP (47).

A surprising finding was that the bovine PARG cDNA clone codes for aprotein of approximately 111 kDa, which is nearly twice the size of thePARG protein isolated from bovine thymus (FIG. 2). It indicates thatPARG contains a protease sensitive site that, following proteolysis,yields a protein fragment of approximately 59 kDa that still retainsenzymatic activity. Several pieces of evidence favor this possibility.(i) Expression of the carboxyl terminal portion of the cDNA resulted inenzymatic activity (FIG. 6, bar 5). (ii) All of the oligopeptidessequenced were located in the carboxyl terminal half of the protein(FIG. 3, FIG. 6 and Table 2). (iii) The only protein, other than 59 kDaprotein detected in the thymus preparation was approximately 111 kD(FIG. 2). (iv) The PARG activity expressed in bacteria was sensitive toproteolysis, yielding a protein of approximately 56 kD (FIG. 6). (v) Thecleavage site in PARG is in the region of the putative NLS and the PARPNLS is located in a protease sensitive site (48). Taken together withthe data suggesting that bovine PARG appears to be coded for by a singlecopy gene (FIG. 7), proteolysis seems likely to explain the presence ofPARG activity of molecular weight of approximately 74 kDa and 59 kDa inbovine thymus preparations (49). Likewise, a similar mechanism couldexplain previous reports of a PARG of 74 kDa isolated from nuclearfractions of guinea pig liver and human placenta (50) and a PARG of 59kDa isolated from postnuclear fractions of guinea pig liver (51).

While proteolysis of a larger protein to yield smaller proteinsretaining PARG activity seems likely to explain the size heterogeneityof PARG previously reported, it remains to be determined if proteolysisnormally occurs in vivo or whether it occurs during purification of theenzyme. While the results presented here show that a full-length proteincan be expressed containing PARG activity (FIG. 8), the molecular sizeof PARG in vivo also remains to be determined. If PARG occurs as alarger protein, an interesting possibility is that the amino terminalregion may be involved in the regulation of enzymatic activity.

Example 5 Expression of bPARG in Escherichia coli

To determine whether the isolated cDNA encoded PARG, bPARG was expressusing two different bacterial expression systems, the pTrcHis XpressSystem™ (Invitrogen, Carlsbad, Calif.), in which the expressed proteincontains a leader polyhistidine sequence, and the glutathioneS-transferasae (GST) gene fusion system (Pharmacia Biotech Inc.,Piscataway, N.J.). For expression in the pTrcHis Xpress system, threedifferent DNA fragments were amplified and inserted into the pTrcHisexpression plasmid. Constructs A and B contained the entire openingreading frame of 110.8 kDa, which together with the fusion partnerpredicted a protein of about 115 kDa. Construct B also contained the3′-untranslated region of the clone. Construct A, containing the cDNAsequence-3 to 2,946, was prepared by subcloning a 2.9kb XhoI-EcoRI DNAfragment amplified from pWL30 with primers WIN34(GCTGCGGGTCTCGACGATGAGTGCGGGC) (SEQ ID NO: 30) and WIN15(GCGTCTAGAATTCACTTGGCTCCTCAGGC) (SEQ ID NO: 31). Construct B, containingthe cDNA sequence-3 to 3,813, was prepared by subcloning a 3.8-kbXhoI-EcoRI DNA fragment amplified from pWL30 with primers WIN34 (SEQ IDNO: 30) and WIN33 (CCGGAATTCGGGTTTTTTGTTAATGAAAATTTATTAAC) (SEQ ID NO:32). Construct C, containing cDNA sequence 964-2,946, was prepared bysubcloning a 2.0-kb DNA fragment amplified from pWL13 with primers WIN14(TCAGAGCAGATGAACTCGAGCAGTCCAGG) (SEQ ID NO: 33) and WIN15 (SEQ ID NO:31). Since the isolated PARG of approximately 59 kDa contained enzymaticactivity, construct C contained only the 75-kDa carboxyl-terminal regionof the PARG, which predicted a fusion protein of approximately 79 kDa.

For expression experiments of bPARG as a GST fusion protein, an insertcontaining the cDNA sequence from position 1138 to 2946 was prepared bysubcloning a 1.8-kb EcoRI-EcoRI fragment amplified from pWL30 with theoligonucleotideCCAATTTGAAGGAGGAATTCCCGCCGCCACCATGAATGATGTGAATGCCAAACGACCTGGA (SEQ IDNO: 34) and WIN15 (SEQ ID NO: 31) as primers. The resulting DNA fragmentwas inserted into the EcoRI site of the pGEX-2T expression vector, andthe plasmid was used to transform E. coli NM522 cells.

For expression experiments, bacterial cultures were grown at 37° C. in1% Bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl to a density ofapproximately 0.6 A₆₀₀(/ml and were induced with 1 mMisopropyl-β-D-thiogalactoside (IPTG). Cells were-collected bycentrifugation, and crude extracts were prepared by sonication (10A₆₀₀/ml) in 10 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl, 0.5mg/ml lysozyme, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.7μg/ml pepstatin A, 0.5 μg/ml leupeptin, and 1 μg/ml aprotinin. Cellextracts were subjected to centrifugation, and the supernatant fractionwas used for assay. PARG assay conditions were as described previously(52). Following incubations, portions of reaction mixture were analyzedby thin layer chromatography or subjected to anion exchange HPLC.

Using a thin layer chromatography assay that measures release of[³²P]ADP-ribose from [³²P]ADP-ribose polymers (53), PARG activity wasdetected in extracts from cells transformed by each of the constructs.FIG. 6 shows results obtained with constructs B and C. Reaction mixturescontained approximately 15,000 cpm of [³²P]ADP-ribose polymers, and thecpm shown represent ADP-ribose released from the ADP-ribose polymers.Bar 1, a strain transformed by pTrcHis without an insert but inducedwith 1 mM IPTG for 5 h at 37° C. A strain containing construct B isshown without the addition of IPTG (bar 2) or after the addition of 1 mMIPTG for 1.5 h (bar 3) or 5 h (bar 4). A strain containing construct C 5h after induction by IPTG is shown in the absence (bar 5) and presence(bar 6) of 167 μm ADP-hydroxymethylpyrrolidine diol (54). No activitywas detected in cells transformed with the empty vector, but activitywas detectable without induction by IPTG, indicating a leaky lacpromoter. The addition of IPTG resulted in a time-dependent increase ofup to approximately 4.5-fold in enzymatic activity. FIG. 6 also showsthat the enzymatic activity was strongly inhibited by the presence ofADP-hydroxymethylpyrrolidine diol, a specific inhibitor of PARG (55).

In FIG. 9, material released from ADP-ribose polymers by anion exchangeHPLC was analyzed. Extracts from a strain containing construct B wereincubated with [³²P]ADP-ribose polymers (56), and a portion was analyzedby anion exchange HPLC as described. The elution times for AMP, ADPR,and PR-AMP are indicated by arrows. The material analyzed was PARGexpressed in E. coli. The results indicated that the material releasedfrom ADP-ribose polymers is exclusively ADP-ribose by strong anionexchange HPLC (FIG. 9), demonstrating that the cell extracts did notcontain any other ADP-ribose polymer-degrading enzymes such asphosphodiesterase, which catalyzes the formation of AMP andphosphoribosyl-AMP (57).

Anion exchange HPLC utilized a Whatman Partisil SAX column equilibratedwith 7 mM potassium phosphate buffer pH 4.0, at a flow rate of 1 ml/min.The sample was diluted in the same buffer, applied to the column, andeluted with a 30-min linear gradient from 7 mM potassium phosphatebuffer, pH 4.0 to 250 mM potassium phosphate buffer, 0.5 M KCl, pH 4.0.

To determine the size of the expressed enzymatic activity, an activitygel assay (58) was used. Activity gel assays for bPARG were done bycasting polyacrylamide gels with automodified PARP containing[³²P]ADP-ribose polymers as described previously (59). Followingelectrophoresis, PARG was renatured by incubating the gels at 25° C. in5 volumes of 50 mM sodium phosphate buffer, pH 7.5, 50 mM NaCl, 10%glycerol, 1% Triton X-100, 10 mM β-mercaptoethanol, changing the bufferevery 3 h for a total of five changes. After an additional incubation at37° C. for 3 h, gels were dried, and PARG activity was detectedfollowing autoradiography as a clear band on a black background. Cellextracts containing PARG fused to GST were examined for binding toglutathione-Sepharose 4B (GSH-Sepharose) (Pharmacia Biotech Inc.)according to the specifications of the manufacturer. No bands wereproduced from extracts from the IPTG-induced pTrcHisB vector that didnot contain an insert. Extracts from cells transformed with a constructcontaining a PARG insert showed bands at approximately 1 15 and 59 kDa(FIG. 8). During storage at 4° C., cell extracts lost activity migratingat the higher molecular weight, while the activity at approximately 59kDa increased.

Expression of bPARG in the pTrcHisB expression vector did not result indetectable amounts of protein by staining the Coomassie Blue. Thus,another construction was designed to overexpress a 69-kDacarboxyl-terminal region of the PARG as a fusion with GST, which allowsconvenient protein purification by affinity chromatography on aGSH-Sepharose column. Two hours after induction with IPTG, strongexpression of a protein migrating at approximately 90 kDa was observed.This protein bound to GSH-Sepharose and was eluted by GSH. The constructcontained a thrombin cleavage site between the GST and the 69-kDa regionof PARG, and the treatment of-the material bound to GSH-Sepharose withthrombin resulted in the release of a protein that migrated atapproximately 59 kDa. This result suggests that the protein purifiedfrom the bovine thymus may be larger than suggested by its migration onSDS-PAGE. The result of this experiment is presented in FIG. 10. Lane 1shows extract from uninduced cells; lane 2 shows extract from cellsinduced with 1 mM IPTG for 2 hours; lane 3 shows proteins in extractsfrom cells shown in lane 2 that bound to GSH-Sepharose; lane 4 showsmaterial released from GSH-Sepharose by treatment with thrombin.

In addition to the GST fusion construct described above, several otherGST fusion proteins have been made. FIG. 11 shows the portions of thebovine PARG gene that have been expressed. The top line represents thestructure of bovine PARG mRNA containing the open reading frame encodingthe 111 kDa PARG protein. The different parts of PARG that have beencloned in expression vectors are represented with the size of theresulting expressed recombinant proteins. The expression of the 65 kDacatalytic domain of PARG (starting at the amino acid MNDV) in pGEX-2T asa fusion protein with glutathione-S-transferase (29 kDa) is detailed.Among the constructs, only the clone designed to express a protein of 69kDa starting at amino acid +380 from the sequence of bovine PARG(bPARC_(MNDV)) allowed high level expression as a fusion protein withglutathione-S transferase (GST). A 1.8 kb PCR EcoRI fragment encodingfor the 65 kDa catalytic domain of PARG was cloned into the EcoRI siteof pGEX-2T giving pGEX-2T-bPARG_(MNDV). This construction results in theexpression of a fused polypeptide consisting of the sequence of GST.Amino acids derived from the polylinker and thrombin site and the 65 kDadomain (FIG. 12).

In addition to various constructs designed to express PARG in E. coli, arecombinant baculovirus expressing a functional PARG has beenconstructed using the methodology of Summers and Smith as set out inU.S. Pat. No. 4,879,236 which is specifically incorporated herein byreference.

bPARG_(MNDV) was cloned in baculovirus transfer vector pVL1393 using theEcoRI site. The recombinant vector was constructed as follows. An insertcontaining the cDNA sequence from position 1138-2946 of bovine PARG wasprepared by subcloning a 1.8 kb EcoRI fragment amplified from pWL30using oligonucleotidesCCAATTTGAAGGAGGAATTCCCGCCGCCACCATGAATGATGTGAATGCCAAACG ACCTGGA (SEQ IDNO: 34) and GCGTCTAGAATTCACTTGGCTCCTCAGGC (SEQ ID NO: 31, WIN15). Theresulting fragment was inserted into the EcoRI site of the pVL1393baculovirus transfer vector. The amplification introduced a Kozakconsensus sequence (gaattcccgccgccaccATGAA SEQ ID NO: 35) at the startsite of translation to enhance expression of the recombinant protein.The resulting recombinant plasmid was cotransfected with linearizeBaculogold™ baculovirus DNA (Pharmingen, San Diego, Calif.) into SF9cells according to the manufacturers instructions. Recombinant virusesisolated using standard techniques. Overexpression of the recombinantprotein was confirmed by Western blot and the results displayed in FIG.13 demonstrate that the 65 kDa domain expressed in E. Coli containedenzymatic activity (lane 2) migrating with the same apparent molecularweight as the enzyme purified from bovine thymus (lane 1). Likewise, aconstruct expressing bPARG_(MNDV) domain in SF9 insect cells infectedwith recombinant baculovirus showed activity (lane 4) migrating with thesame apparent molecular weight.

Example 6 Northern Blot Analysis

An surprising feature of the consensus full-length cDNA clone was thatit predicted expression of a protein of approximately 111 kDa (FIG. 3,SEQ ID NO: 1, and SEQ ID NO: 2), while the enzymatically active PARGfrom thymus had a molecular weight of approximately 59 kDa (FIG. 2). Todetermine the size of the RNA transcript for PARG, total RNA andpoly(A)+ RNA were isolated from bovine kidney (MDBK) cells and annealedusing Clone 4 as the hybridization probe.

Total cytoplasmic RNA and poly(A)+ RNA were isolated from bovine kidneyMDBK cells (ATCC #CCL22) using TRIzol reagent (Gibco/BRL) following themanufacturer's recommendations. After the RNA was fractionated, it wasthen transferred to nylon membranes and hybridized with Clone 4 (FIG. 3)radiolabeled by a random hexamer priming method (21). The results arepresented in FIG. 4. Total RNA (5 μg, lanes 1A and 1B) and poly(A)+ RNA(4 μg, lanes 2A and 2B) were separated on a denaturing agarose gel (60).Panel A shows the ethidium bromide stained gel and panel B shows theautoradiogram of a Northern blot analysis using a random primed,³²P-labeled DNA probe constructed from Clone 4 (FIG. 3). A singletranscript of approximately 4.3 kb was detected in the poly(A)+ RNA(FIG. 4, lane 2). Thus, the transcript size was consistent with theexpression of a 111 kDa PARG protein.

Example 7 Southern Blot Analysis of PARG Genomic Complexity

Previous studies have reported that PARG isolated from nuclear fractionshad a molecular weight of approximately 75 kDa (61), while PARG isolatedfrom whole cell homogenates or postnuclear supernatant fractions had amolecular weight of approximately 59 kDa (62). These results suggestthat either two or more genes may code for PARG or that proteolysisgenerates lower molecular weight forms from higher molecular weightforms. The cDNA isolated encoded a protein considerably larger than anyPARG proteins previously described, consistent with the possibility thatthe different forms of PARG are derived from a single form byproteolytic cleavage. To test the hypothesis that PARG is encoded by asingle copy gene, the genomic complexity of the PARG gene was analyzedby a Southern hybridization experiment.

Total genomic DNA was prepared from bovine thymus tissue as describedpreviously (63) and DNA (10 μg) was digested with EcoRI, BglII, XbaI orPstI, fractionated on a 1% agarose gel, transferred to a nylon membrane(Hybond N+, Amersham), and hybridized using an 828 bp HindIII fragmentof Clone 1 radiolabeled as described for clone 4 above (64).Pre-hybridizations and hybridizations were carried out at 42° C. in 50%fornamide, 0.25 M sodium phosphate buffer, pH 7.2, 0.25 M NaCl, 7% SDS,1 nM EDTA. The blot was annealed with a ³P-labeled DNA probecorresponding to the carboxyl terminal region of the PARG protein.

The results of the Southern blot analysis are presented in FIG. 7.Genomic DNA was digested with four different restriction enzymes, EcoRI(lane 1), BglII (lane 2), XbaI (lane 3) and PstI (lane 4), none of whichcleave within the carboxyl terminal region of the PARG cDNA. Followingelectrophoresis, the restriction digests were subjected to hybridizationwith a probe that corresponded to the carboxyl terminal region of thePARG cDNA. The analysis displayed in FIG. 7 shows that, in eachrestriction digest, the probe hybridized primarily with a singlerestriction fragment. The fainter signals likely reflect the presence ofintrons in the PARG gene. This result indicates that PARG is encoded bya single copy gene in the bovine genome.

Example 8 Isolation and Characterization of PARGs from Other Species

The isolation and characterization of bovine cDNA encodingpoly(ADP-ribose) glycohydrolase (PARG) has been described above. Usingthe information provided by the sequencing of bovine PARG, various toolswere used, including public sequence databases searches and screening ofcDNA libraries using PARG specific probes, to clone and sequence thecDNA and determine the primary structure of PARG from human, mouse,Drosophila and Caernorhabditis elegans. Mammalian sequences newlyobtained using this combined strategy show high sequence similarity tobovine PARG (bPARG), whereas the sequences of Drosophila and C. elegansonly display significant homologies in the region responsible of thecatalytic activity of the protein.

The strategy followed to obtain cDNAs coding for proteins with sequencesimilarity to bovine PARG is summarized in FIG. 14. dBEST, GenBank,SwissProt and PIR databases were searched for PARG like-sequences at thenucleotide or amino acid level using the programs BLASTn, TBLASTn(Altschul et al., 1990) respectively, available at the NIH site on theWorldwide Web, and also included in the sequence analysis package fromthe Genetic Computer Group, Inc. (GCG) (Madison, Wis.), version 9.1.Both programs perform pair-wise sequence comparisons on multiplenucleotide or amino acid sequences. PARG multiple sequence comparisonsobtained with these programs are very similar. Box-shading of the aminoacids in the multi-sequence alignment was obtained using the programBOXSHADE (K. Hofmann and M. D. Baron). The first step involved extensivesearching for sequences with bPARG similarity in various databases. As aresult of this search several partial nucleotide sequences sharingextensive homologies with bPARG cDNA were obtained from the dBESTdatabase (65). These sequences were the result of random cloning andsequencing of partial cDNAs clones obtained from mRNAs expressed invarious tissues and organisms. Among them, partial cDNAs coding for PARGfrom human and mouse were available. One of these human clones wasparticularly interesting as its sequence (2500 bp long) overlapped thecoding sequence of bovine PARG from aa470 to aa977 (Carboxy terminusend) and contained all the 3′ untranslated region of the human PARGcDNA. This clone (No. 50859; GenBank accession number: H 17209) wasrequested and freely obtained from the IMAGE Consortium (incollaboration with Washington University School of Medicine in St.Louis, Mo. and Merck & Co., info@image.llnl.gov). The sequence of theclone was then completed. This partial cDNA permitted design of aradiolabeled probe (fragment HindIII—KpnI of 677 bp) specific to humanPARG (SEQ ID NO: 36).

Example 9 Cloning and Sequencing

The cloning procedures used in this work generally known and are alsodescribed in details in the book, Molecular Cloning: A Laboratory Manual(Maniatis et al., 1982). DNA sequencing was performed using thedideoxynucleotide method of Sanger (Sanger et al., 1977). Chemicalreagents were purchased from Sigma (St. Louis, Mo.). Restrictionenzymes, T4 DNA ligase were from New England Biolabs, Inc. (Beverly,Mass.), T7 DNA polymerase Sequenase from US Biochemical (Cleveland,Ohio), Calf Intestine Phosphatase from Boehringer, Manheim(Indianapolis, Ind.). The phagemid pTZ18/19R is from Pharmacia(Piscataway, N.J.). The labeled nucleotides α-[³⁵S]-dATP andα-[³²P]-dCTP were purchased from ICN (Costa Mesa, Calif.). Human thymusand murine liver 5′-stretch cDNA libraries cloned in the vector λgt 10were from Clontech (Palo Alto, Calif.).

A single, isolated colony of C600Hfl E. coli strain was picked and grownin 5 ml of Luria-Bertani medium (LB)+10 mM MgSO₄+0.2% maltose ovemightat 37° C. in a shaker. The bovine library lysate was diluted 1:250,000and incubated with the C600Hfl bacterial overnight culture and 1× lambdadilution buffer. Next, LB soft top agar+10 mM MgSO₄ was added, and theentire mixture was quickly poured onto 90 mm LB agar+10 mM MgSO₄ plates.The plates were cooled briefly at room temperature to allow the inoculumto soak into the agar before they were incubated at 37° C. for 6-7 hr.The number of clear plaques was counted to determine the titer.

Plaques containing the entire library that had been plated weretransferred to nitrocellulose or nylon membranes. The filters were thenwashed in a 1.5 M NaCl/0.5 M NaOH solution to lyse the cells. This wasfollowed by a 5 min wash in neutralizing solution (1.5 M NaCl/1 M Trisbuffer pH 8). Finally, the filters were rinsed in 0.2×SSPE (30 mM NaCl/2mM sodium phosphate buffer pH 7.2/0.2 mM EDTA) (Sambrook et al., 1992).The filters were then dried and baked in a 80° C. oven for 2 hr to fixthe lysed plaques onto the filters.

Radioactive probes were prepared using a random hexamer priming method.Pre-hybridizations and hybridizations were carried out at 42° C. in 50%formamide, 0.25 M sodium phosphate buffer, pH 7.2, 0.25 M NaCl, 7% SDS,1 mM EDTA.

Example 10 Specific Methods used for Library Screening

All the cloning procedures used in obtaining the additional PARG cDNAsand determining their sequences were performed essentially as describedfor the bovine PARG cDNA and sequence. Human thymus and murine liver5′-stretch cDNA libraries cloned in the vector λgt 10 were from Clontech(Palo Alto, Calif.).

Library plating and titering: A single, isolated colony of C600Hfl E.coli strain was picked and grown in 5 ml of Luria-Bertani medium (LB)+10mM MgSO4+0.2% maltose overnight at 37° C. in a shaker. The librarylysate was diluted 1:250000 and incubated with the C600Nfl bacterialovernight culture and 1× lambda dilution buffer. Next, LB soft topagar+10 mM MgSO4 was added, and the entire mixture was quickly pouredonto 90 mm LB agar+10 mM MgSO4 plates. The plates were cooled briefly atroom temperature to allow the inoculum to soak into the agar before theywere incubated at 37° C. for 6-7 hr. The number of clear plaques wascounted to determine the titer.

Plaque lifts: Plaques containing the entire library that have beenplated are transferred to nitrocellulose or nylon membranes. The filtersare then washed in a 1.5 M NaCl/0.5 M NaOH solution to lyse the cells.This is followed by a 5 min wash in neutralizing solution (1.5 M NaCl/1M Tris buffer pH 8). Finally, the filters are rinsed in 0.2×SSPE (30 mMNaCl/2 mM sodium phosphate buffer pH 7.2/0.2 mM EDTA) (66). The filtersare then dried and baked in a 80° C. oven for 2 hr to fix the lysedplaques onto the filters.

Making a radioactive probe and Hybridizations: Radioactive probes wereprepared using a random hexamer priming method. Pre-hybridizations andhybridizations were carried out at 42° C. in 50% formrnamide, 0.25 Msodium phosphate buffer, pH 7.2, 0.25 M NaCl, 7% SDS, 1 mM EDTA. Thispartial cDNA allowed to design a radiolabeled probe (fragmentHindIII—KpnI of 750 bp long) specific to human PARG.

Example 11 Screening of a Human Thymus 5′-Stretch cDNA Libraries

Multiple screenings of a human thymus 5′-stretch cDNA library wereperformed to complete the cloning of human PARG cDNA. For each screeninga new probe was designed and used to screen approximately one millionrecombinants of the library. During each round of screening, overlappingclones were isolated at high stringency conditions and subcloned intothe EcoRI site of pTZ18/19R phagemid using standard techniques. Thedifferent positive clones (J5, C, E1, E2, M, M′, M″, P′, P″, Of, 02)were characterized by restriction analysis, subcloned into theappropriate restriction sites of pTZ18/19R as necessary and sequenced inboth strand using the dideoxynucleotide method. The probe used tocomplete the cloning of the human cDNA library is shown is SEQ ID NO:37. Finally, a full-length cDNA sequence was assembled which encodes thehuman PARG. The sequence of the cDNA encoding human PARG is presented inthe sequence listing as SEQ ID NO: 3 and the amino acid sequence ofhuman PARG is presented in the sequence listing as SEQ ID NO: 4.

The human PARG sequence shares extensive amino acid sequence homologieswith bovine PARG with more than 89% identity. The sequence similarity isalso high at the nucleotide level particularly in the region coding forthe protein (174ATG-TGA3104). Surprisingly the 5′-untranslated region ofthe human sequence displays a completely different sequence with anextensive sequence similarity with highly repeated polymorphic DNAsequences found in the human genome such as Alu repetitive elements orvariable number of tandem repeats (VNTR).

Example 12 Screening of Mouse Liver 5′-Stretch cDNA Libraries

To isolate a PARG cDNA from the mouse liver cDNA library, a probe wasdesigned from the human cDNA clone coding for PARG. Analysis of thebovine and human sequences revealed that PARG was highly conservedbetween these two species, suggesting that it might also be conserved inthe mouse. Based on the restriction map of the human cDNA clone, aregion in the human clone was selected, located where the active site ofthe protein is encoded, that exhibited near identity to its counterpartin the bovine clone. This region, consisting of approximately 800 bases,was excised from the entire human clone by digestion with therestriction endonuclease, HindIII, then purified by agarose gelseparation and radiolabeled by random priming.

This probe was used to screen a mouse liver 5′-stretch cDNA library. Oneclone consistently hybridized with the probe. After two rounds ofscreening to ensure the purity of the clone the 2.5 kb insert wassubcloned into the plasmid pTZ19R and sequenced. Comparison with thesequence of bovine and human PARG showed that this clone had the partialsequence that has extensive similarities to the two other mammaliansequences covering almost entirely the coding region from nucleotide −10to a few nucleotides from the end of the coding region. A second screenwas performed to obtain the missing part of the cDNA using a radioactiveprobe specifically designed to hybridize with the region the most 3′ ofthe previous clone to increase the chance to get the missing part of thecDNA.

With this new probe, the same mouse liver cDNA library was screened toobtain a second clone, containing an insert that was about 3 kb. Thisclone was purified, subcloned and sequenced. The sequence showed thatthis second clone starts at amino acids 634, extends toward the stopcodon to approximately 900 nucleotides into the 3′ non-coding region.

A search of the dBEST database turned up one significant match to a 400bp fragment cloned from mouse muscularis. This fragment had an exactmatch to the very tail end of the second clone and exceeded it by 34bases. This extra extension contained the oligo A sequence as well asthe polyadenylation signal. Because there was an exact match, the cDNAsequence was completed using this information coming from the database.The complete cDNA sequence of murine PARG is presented in the SequenceListing as SEQ ID NO: 5 and SEQ ID NO: 6.

Example 13 Obtaining the Drosophila PARG cDNA

Among the clones obtained from DNA databases searches were severalclones from the Drosophila genome sequencing project (EuropeanDrosophila Genome Sequencing Consortium) as well as the Drosophilaexpression sequence TAG sequencing project (67). The EST clone wasrequested from the University of California Berkeley and obtained.Because the sequence published in the dBEST database was only partial,its sequence was completed in our laboratory and compared to a genomicsequence, part of the distal X chromosome of Drosophila melanogastersubmitted by Murphy et al., August 1997 which presumably contains thegene of Drosophila PARG. The 768 aa shares less homologies with only 40%identity (48% similarity) mainly located in the catalytic domain of theprotein. The domain organization of the protein is also very differentwith an unknown domain of 20 kDa located Carboxy terminus of the highlyconserved active domain. (See FIG. 15). The sequence of the cDNAencoding the Drosophila PARG is presented in the Sequence Listing as SEQID NO: 7 and the amino acid sequence of the Drosophila PARG is presentedin the Sequence Listing as SEQ ID NO: 8.

Example 14 Obtaining the C. elegans PARG Sequence

This sequence has been obtained by searching the GenBank database withthe mammalian PARG protein sequence. A sequence with PARG similarity wasfound in the cosmid F20C5 (Accession number: Z68161, SEQ ID NO: 38)derived from the C. elegans genomic DNA (68). The overall sequenceconservation (726aa, MW 83129 Da) with the other PARG sequences is asfollows: 32% similarity and 22% identity with the mammalian PARG and 39%similarity and 30% identity with the Drosophila PARG. The sequence ispresented in the Sequence Listing as SEQ ID NO: 38 (Genbank accessionnumber CEF20C5). SEQ ID NO: 38 contains 12 exons as follows: exon 1 from3591 to 3635; exon 2 from 3681 to 4121; exon 3 from 5065 to 5235; exon 4from 5930 to 6152; exon 5 from 6200 to 6267; exon 6 from 7246 to 7338;exon 7 from 7386 to 7553; exon 8 from 7738 to 7853; exon 9 from 8153 to8435; exon 10 from 8487 to: 8610; exon 11 from 8662 to 8952; and exon 12from 9383 to: 9540. The coding sequence of the CePARG protein, which ispublicly available from Accession number: Z68161, is referred to in theSequence Listing as SEQ ID NO: 9. Its corresponding amino acid sequenceis referred to in the Sequence Listing as SEQ ID NO: 10. The amino acidsequence of the C. elegans PARG is presented on the alignment (FIG. 16)

Example 15 Cloning and Overproduction of the Carboxyl-Terminus 69 kDaDomain of Bovine PARG (bPARG) in E. coli

As described, above, bovine PARG is encoded by a messenger of 4 kbpredicting a protein of 110 kDa, almost twice the size of the purifiedenzyme (65 kDa). It is also demonstrated that bPARG can be expressed inE. coli as an active enzyme either as a 110 kDa or a 65 kDa protein.This result combined with other evidence implies that the active site ofPARG is located in the carboxyl-terminal part of the protein. FIG. 11 isa schematic representation of the different clones we have expressed inbacteria. Among them, only the clone designed to express a protein of 69kDa starting at the amino acid +380 from the sequence of bovine PARG(bPARG_(MNDV)) allowed high level expression as a fusion protein withglutathione-S transferase (GST).

The heterologous expression of bPARG_(MNDV) was conducted as representedin FIG. 12. The 1.8 kb cDNA encoding the 69 kDa carboxyl-terminal partof bovine PARG was amplified by PCR and cloned in the EcoRI site-ofpGEX-2T vector (Pharmacia) in fusion with GST giving thepGEX-2T-bPARG_(MNDV) plasmid. E. coli NM522 cells transformed with thepGEX-2T-bPARG_(MNDV) were induced by addition of IPTG, resulting inexpression of a 90 kDa fusion protein. The fusion protein can beconveniently purified using Glutathione-Sepharose and the bPARG_(MNDV)can be released by treatment with thrombin while the GST protein remainsbound to the beads of GSH-Sepharose. In this manner milligram amounts ofprotein can be routinely obtained.

Example 16 Characterization of the Purified 65 kDa Domain and theGeneration of Antibodies

The purified bPARG_(MNDV) was characterized by activity gel assays (69)by casting polyacrylamide gels with automodified PARP containing[³²P]ADP-ribose polymers. The results demonstrate that the 65 kDa domainexpressed in E. coli contained enzymatic activity migrating with thesame apparent molecular weight as the enzyme purified from bovinethymus. Likewise, a construction expressing bPARG_(MNDV) domain in SF9insect cells infected with recombinant baculovirus showed activitymigrating with the same apparent molecular weight.

The availability of PARG cDNA allows the development of new moleculartools to study this enzyme in its cellular context. Until this work, itwas not possible to obtain PARG in sufficient quantities to produceantibodies against the protein. The antibody raised against bovine PARGis able to recognize PARG from other organisms and, thus, will bevaluable in characterizing PARG in vivo under defined physiologicalconditions in many different organisms.

Antibodies against bPARG_(MNDV) overexpressed in E. coli were raised inrabbits using the procedure described by Vaitukaitis (70). Specific highaffinity antibodies are generated by administration of small doses ofimmunogens intradermally over a wide anatomic area of the animal.Rabbits were immunized by three injections of 10-50 μg of the Mr 65,000protein band excised from a preparative SDS polyacrylamide gel. Titerand affinity of sera harvested weekly were followed by conventionalmethods. Peak affinity was attained in 8 to 10 weeks after primaryimmunization. For each animal, a preimmune serum was retained as acontrol.

FIG. 17 shows a Western blot experiment demonstrating the specificity ofthe resulting. PARG anti-serum against the purified bPARG from thymus(lane 1), SF9 protein extract expressing 65 kDa-bPARG_(MNDV) inrecombinant baculovirus (lane 2), recombinant 65 kDa-PARG_(MNDV)purified by treatment with thrombin from GSH-Sepharose (lane 3), and anE. coli crude extract expressing the fusion protein GST-65kDa-PARG_(MNDV) (lane 4). The pre-immune serum did not show reactivityagainst any of these fractions even at a low dilution ( 1/250).

Antibodies directed against the 45 kDa -terminal have also beengenerated using the same strategy used to generate antibodies againstthe catalytic domain. This involved the overexpression of the 45 kDaprotein domain in E. coli in a construct designed for easy purification,followed by injection of the purified protein into rabbits. Theheterologous expression of PARG45 was conducted by cloning a part (1.1kb) of the coding region of the cDNA, generated by PCR amplification ofthe region located between the ATG(267) codon and nucleotide 1400 in thebovine sequence, into the Eco RI site of the bacterial expression vectorpGEX-2T (Pharmacia) in fusion with glutathione-S-transferase. E. coliNM522 cells transformed with this construct were induced by addition ofIPTG, resulting in expression of a 72 kDa fusion protein. The fusionprotein was purified using glutathione Sepharose and the PARG45 wasreleased by treatment with thrombin, while the GST protein remainedbound to the GSH Sepharose beads. In this manner milligram amounts ofprotein were obtained. Antibodies against PARG45 overexpressed in E.coli were raised in rabbits using the procedure described (71). Specifichigh affinity antibodies were generated by administration of small dosesof immunogens subcutaneously over a wide area of the animal. Rabbitswere immunized by three injections of 10-50 μg of the 45 kDa proteinband excised from a preparative SDS-polyacrylamide gel. Titer andaffinity of sera harvested weekly were followed by conventional methods.Peak affinity was attained in 8 to 10 weeks after primary immunization.For each animal, a preimmune serum was retained as a control.

Example 17 Conservation of PARG in Tissues and Organisms

Tissue and cell extracts from different origins were homogenized in acold hypotonic lysis buffer containing a cocktail of protease inhibitorsand sonicated. SDS and β-mercaptoethanol were added to insureinactivation of any remaining active proteases. Thirty μg of proteinfrom each extract was analyzed by Western-blot using the anti-PARGantibody (FIG. 18). In all of the fractions from bovine tissues, PARGwas observed as a major band at 65 kDa. However, less intense, discreteproteins of higher molecular weight were also detected. These proteinsmay correspond to different forms of PARG; the band of highest molecularweight (about 115 kDa) found in thymus extract likely corresponds to thefull-length of PARG (111 kDa) as deduced from the cDNA. Multiple specieswere detected in cell extracts from mouse fibroblasts, rat PC 12 cells,and SF9 insect cells. This result shows that the sequence of PARG iswell conserved phylogenetically. Moreover, the conservation includesmultiple molecular forms of the protein.

Example 18 Regulation of the Expression of PARG

In the metabolism of ADP-ribose polymers, the activities of PARP andPARG are closely related. Soon after polymer has been synthesized byPARP following DNA damage, it is extensively degraded by PARG. The netresult is that the polymer has a very short half life. The closerelationship between the two proteins suggests a possible mode ofregulation in which PARG expression depends on the presence of PARP. Inorder to test if the presence or the absence of PARP influences theexpression of PARG, a Western Blot experiment was performed with cellextracts from mouse fibroblasts of different PARP genotypes (72).

Cell extract (30 μg) from mouse cells with PARP+/+, PARP± and PARP−/−genotypes were separated by SDS-PAGE, transferred to a membrane andprobed with the antibodies indicated. The results are shown in FIG. 19.In FIG. 19, purified PARG (50 ng) from bovine thymus (lane 1), 30 μg ofprotein of a total extract from PARG recombinant baculovirus infectedSF9 cells (lane 2), 150 ng of purified recombinant PARG produced in thebacteria (lane 3) and 30 μg of protein of a crude extract from E. coliNM522 transformed with pGEX-2T-bPARG_(MNDV) 2 h after induction by IPTG(lane 4) were separated on a 0.1% SDS-12% polyacrylamide gel, thentransferred on nitrocellulose, and incubated with a 1/5000 dilution ofthe rabbit polyclonal antiserum raised against the 65 kDa domain ofbPARG. Proteins were revealed by immunofluorescence with the ECLdetection kit (Amersham) and autoradiography. Panel A is a western blotof PARP in cells of varying PARP genotype showing the results of theanalysis using anti-PARP antibodies. The amount of PARP expressed variesas expected dependant upon the genotype of the cell line with thePARP−/− cell line producing no detectable amount of PARP. Panel B iswestern blot of PARG from various tissues using an anti-PARG antibody.It shows that the level of PARP is variable. The amount of PARG presentin the cell extracts was not dependent upon the PARG genotype of thecell. Further support for this view is provided by the results of thePARG activity assay presented in panel C. The specific activity of PARGdetected in the extracts showed no significant difference among thethree genotypes.

Example 19 Preparation PARG Gene Ablation (Knockout) Animals

One embodiment of the present invention is experimental animals withtargeted mutations in the PARG gene. These animals may be constructedusing standard techniques and the cDNA sequence of the PARG. In thefollowing example, a mouse containing a targeted mutation in the PARGgene is constructed. Those skilled in the art will readily appreciatethat other experimental animals, including but not limited to rats,guinea pigs, hamsters and the like, may be constructed using similartechniques. The construction of animals with disrupted genes may beaccomplished using standard techniques such as those described byMoreadith (73). Further, cells lines, construction kits, and protocolsfor knockout mice are available from commercial suppliers such asStratagene (Stratagene 1999 catalog, La Jolla, Calif.). Commercialservices such as Lexicon Genetics (The Woodlands, Tex.) and ChrysalisDNX Transgenic Sciences (Princeton, N.J.) also offer complete ES cellknockout mice production services.

A genomic clone of the murine PARG enzyme may be isolated from a genomiclibrary by screening with a probe derived from the. cDNA sequence ofPARG. A mouse 129/SV genomic library (Stratagene) containing mousegenomic sequences in λ phage was screened using a 2.49 kb fragment ofthe mouse PARG cDNA as a probe. A partial restriction map of onepositive clone thus isolated, R1, is provided in FIG. 20. The R1 clonecontains the genomic sequence corresponding to the 5 ′-most end of themurine cDNA. The clone was subcloned into pBluescript as threefragments. The plasmid containing the 5′-end contained a 2.8 kbNotI-EcoRI fragment and was designated p2.8R. The fragment containingthe central portion contained a 3.5 kb EcoRI fragment and was designatedp3.5R. The plasmid containing the 3′-end of the gene contained a 7.0 kbEcoRI-NotI fragment and was designated 7.0R. Sequencing the resultingplasmids revealed that p2.8R contained no sequences corresponding to thecDNA, p3.5R contains a 1.5 kb promoter region and/or untranslated regionand exon I coding for 72 amino acids including the initiation ATG codonand p7.0R contains at least 4 additional exons. Gene targeting vectorsmay be constructed using both p3.5R and p7.0R.

A gene targeting vector may contain one or more selection genes flankedby genomic sequences. The targeting vector is introduced into the genomeby homologous recombination resulting in the incorporation of theselection gene into the genome of the cell. The mouse PARG gene wastargeted using a “conditional” inactivation procedure outlined in FIG.21. This approach allows the production of viable animals even if thedisrupted gene results in a lethal phenotype since the gene is notdisrupted until a second “conditional” recombination event is induced.

A lox-P sequence may be inserted into the first intron. A cassetteexpressing the neomycin resistance gene (neo) and the thymidine kinasegene (TK) flanked by two additional lox-P sites may be placed in intron2. In the presence of Cre recombinase, recombination will occur betweentwo lox-P sites thereby deleting the genomic sequences present betweenthe sites. A MC1-DTA cassette is ligated at the 3′-end of the vector toreduce random integration of the vector into the genome.

The targeting vector may be introduced into embryonic stem cells by anymethod known to those skilled in the art such as transfection,lipofection or electroporation. In a preferred embodiment, the targetingvector will be introduced into embryonic stem cells by electroporation.After homologous recombination, cells containing the neo gene will beselected for using G418. Selected cells will then be analyzed by PCR andSouthern blot.

To generate mutant alleles of PARG, the positive embryonic stem cellclones identified will be transfected with a plasmid expressing Crerecombinase. The action of Cre recombinase will result in threedifferent mutant alleles. Mutant allele I contains a deletion in exon 2but still maintains the selection genes neo and TK. Mutant allele IIcontains the genomic sequence for exon 2 flanked by two lox-P sites(exon 2 is said to be “floxed”) and does not contain the selectiongenes. Mutant allele III has a deletion of the genomic sequences anddoes not contain the selection genes.

Mice containing each of the three mutant alleles may be constructed bymicroinjecting embryonic stem cells containing the mutant allele intoblastocytes resulting in the production of chimeric and mutant mice.Mice homozygous in mutant allele I or III will be null mutants in thatthey will be unable to express a functional PARG enzyme due to the lossof required genomic sequences. In the absence of Cre recombinase, micecontaining mutant allele II will express a wild type protein. In thepresence of the Cre recombinase, the PARG will lose exon 2, thusproducing an inactive protein. To inactivate the gene, these mice willbe bred to mice expressing Cre recombinase under the control of a tissuespecific promoter. This will result in mice expressing PARG in sometissues and not expressing PARG in others. Mice homozygous in mutantallele II, will be valuable for evaluating the role of PARG in specifictissues.

Although the present invention has been described with reference tocertain examples for purposes of clarification and illustration. Itshould be appreciated that certain obvious improvements andmodifications can be practiced within the scope of the appended claimsand their equivalents. Other embodiments and uses of the invention willbe apparent to those skilled in the art from consideration of thespecification and practice of the invention disclosed herein. All U.S.patents GenBank sequence listings, and other references noted herein forwhatever reason are specifically incorporated by reference. Thespecification and examples should be considered exemplary only with thetrue scope and spirit of the invention indicated by the followingclaims.

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1-42. (canceled)
 43. A method of screening candidate molecules for PARGmodulating activity, comprising the steps of: providing a purified PARGenzyme; assaying the enzyme in the presence of a candidate molecule tobe screened; and comparing the activity of the PARG enzyme in thepresence of the molecule to the activity of the PARG enzyme in theabsence of the molecule. 44.-66. (canceled)